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


Wednesday, September 30th, 2015

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Moncrieff, S., Moncan, M., Scialpi, F. and Ditzel, M. (2015). Regulation of Hedgehog ligand expression by the N-end rule ubiquitin-protein ligase Hyperplastic discs and the Drosophila GSK3β homologue, Shaggy. PLoS One 10: e0136760. PubMed ID: 26334301
Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. This study revealed that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, Hyd esd found to physically interact with Sgg and Ci. Taken together it iw proposed that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, these Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals.

Rice, C., Beekman, D., Liu, L. and Erives, A. (2015). The nature, extent, and consequences of genetic variation in the opa repeats of Notch in Drosophila. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26362765
Polyglutamine (pQ) tracts are abundant in proteins co-interacting on DNA. The lengths of these pQ tracts can modulate their interaction strengths. However, pQ tracts > 40 residues are pathologically prone to amyloidogenic self-assembly. This study assesses the extent and consequences of variation in the pQ-encoding opa repeats of Notch in Drosophila melanogaster. Sanger sequencing was used to genotype opa sequences (5'-CAX repeats), which have resisted assembly using short sequence reads. While most sampled lines carry the major allele opa31 encoding Q13HQ17 or the opa32 allele encoding Q13HQ18, many lines carry rare alleles encoding pQ tracts > 32 residues: opa33a (Q14HQ18), opa33b (Q15HQ17), opa34 (Q16HQ17), opa35a1/opa35a2 (Q13HQ21), opa36 (Q13HQ22), and opa37 (Q13HQ23). Only one rare allele encodes a tract < 31 residues: opa23 (Q13-Q10). This opa23 allele shortens the pQ tract while simultaneously eliminating the interrupting histidine. The study introgressed these opa variant alleles into common backgrounds, and measured the frequency of Notch-type phenotypes. Homozygotes for the short and long opa alleles have defects in embryonic survival and sensory bristle organ patterning, and sometimes show wing notching. Consistent with functional differences between Notch opa variants, it was found that a scute inversion carrying the rare opa33b allele suppresses the bristle patterning defect caused by achaete/scute insufficiency, while an equivalent scute inversion carrying opa31 manifests the patterning defect. These results demonstrate the existence of potent pQ variants of Notch, and the need for long read genotyping of key repeat variables underlying gene regulatory networks.

Surabhi, S., Tripathi, B.K., Maurya, B., Bhaskar, P.K., Mukherjee, A. and Mutsuddi, M. (2015). Regulation of Notch signalling by an evolutionary conserved DEAD Box RNA helicase, Maheshvara in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 26400611
Notch signalling is fine-tuned at multiple levels and mis-regulation of Notch has been implicated in a variety of human diseases. This study characterized maheshvara (mahe), a novel gene in Drosophila which encodes a putative DEAD box protein that is highly conserved across taxa and belongs to the largest group of RNA helicase. Dynamic pattern of mahe expression along with the maternal accumulation of its transcripts is seen during early stages of embryogenesis. In addition, a strong expression is also seen in the developing nervous system. Ectopic expression of mahe results in a variety of defects, many of which resemble typical Notch loss-of-function phenotype. Ectopic expression of mahe in the wing imaginal discs leads to loss of Notch targets, Cut and Wingless. Interestingly, Notch protein levels are also lowered, whereas no obvious change is seen in the levels of Notch transcripts. In addition, mahe overexpression can significantly rescue ectopic Notch mediated proliferation of eye tissue. Further, mahe genetically interacts with Notch and its cytoplasmic regulator deltex in trans-heterozygous combination. Taken together, the study reports identification of a novel evolutionary conserved RNA helicase mahe, which plays a vital role in regulation of Notch signalling.

Sawala, A., Scarcia, M., Sutcliffe, C., Wilcockson, S. G. and Ashe, H. L. (2015). Peak BMP responses in the Drosophila embryo are dependent on the activation of integrin signaling. Cell Rep 12: 1584-1593. PubMed ID: 26321638
Within a 3D tissue, cells need to integrate signals from growth factors, such as BMPs, and the extracellular matrix (ECM) to coordinate growth and differentiation. This study used the Drosophila embryo as a model to investigate how BMP responses are influenced by a cell's local ECM environment. Integrins (see Myospheroid), which are ECM receptors, were shown to be absolutely required for peak BMP signaling. This stimulatory effect of integrins requires their intracellular signaling function, which is activated by the ECM protein collagen IV. Mechanistically, integrins interact with the BMP receptor Thickveins and stimulate phosphorylation of the downstream Mad transcription factor. The BMP-pathway-enhancing function of integrins is independent of focal adhesion kinase, but it requires conserved NPXY motifs in the β-integrin cytoplasmic tail. Furthermore, it was shown that an α-integrin subunit is a BMP target gene, identifying positive feedback between integrin signaling and BMP pathway activity that may contribute to robust cell fate decisions.

Tuesday, September 29th

Kim, H.J., Ahn, H.J., Lee, S., Kim, J.H., Park, J., Jeon, S.H. and Kim, S.H. (2015). Intrinsic dorsoventral patterning and extrinsic EGFR signaling genes control glial cell development in the Drosophila nervous system. Neuroscience [Epub ahead of print]. PubMed ID: 26318336
Dorsoventral patterning and EGFR signaling genes are essential for determining neural identity and differentiation of the Drosophila nervous system. Their role in glial cell development in the Drosophila nervous system is not clearly established. This study demonstrates that the dorsoventral patterning genes, vnd, ind, and msh, are intrinsically essential for the proper expression of a master glial cell regulator, gcm, and a differentiation gene, repo, in the lateral glia. In addition, it was shown that esg is particularly required for their expression in the peripheral glia. These results indicate that the dorsoventral patterning and EGFR signaling genes are essential for identity determination and differentiation of the lateral glia by regulating proper expression of gcm and repo in the lateral glia from the early glial development. In contrast, overexpression of vnd, msh, spi, and Egfr genes repress the expression of Repo in the ventral neuroectoderm, indicating that maintenance of correct columnar identity along the dorsoventral axis by proper expression of these genes is essential for restrictive formation of glial precursor cells in the lateral neuroectoderm. Therefore, the dorsoventral patterning and EGFR signaling genes play essential roles in correct identity determination and differentiation of lateral glia in the Drosophila nervous system.

Yalgin, C., Ebrahimi, S., Delandre, C., Yoong, L. F., Akimoto, S., Tran, H., Amikura, R., Spokony, R., Torben-Nielsen, B., White, K. P. and Moore, A. W. (2015). Centrosomin represses dendrite branching by orienting microtubule nucleation. Nat Neurosci. PubMed ID: 26322925
Neuronal dendrite branching is fundamental for building nervous systems. Branch formation is genetically encoded by transcriptional programs to create dendrite arbor morphological diversity for complex neuronal functions. In Drosophila sensory neurons, the transcription factor Abrupt represses branching via an unknown effector pathway. Targeted screening for branching-control effectors identified Centrosomin, the primary centrosome-associated protein for mitotic spindle maturation. Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization.

Dascenco, D., Erfurth, M. L., Izadifar, A., Song, M., Sachse, S., Bortnick, R., Urwyler, O., Petrovic, M., Ayaz, D., He, H., Kise, Y., Thomas, F., Kidd, T. and Schmucker, D. (2015). Slit and Receptor tyrosine phosphatase 69D confer spatial specificity to axon branching via Dscam1. Cell 162: 1140-1154. PubMed ID: 26317474
Axonal branching contributes substantially to neuronal circuit complexity. Studies in Drosophila have shown that loss of Dscam1 receptor diversity can fully block axon branching in mechanosensory neurons. This study reports that cell-autonomous loss of the receptor tyrosine phosphatase 69D (RPTP69D) and loss of midline-localized Slit inhibit formation of specific axon collaterals through modulation of Dscam1 activity. Genetic and biochemical data support a model in which direct binding of Slit to Dscam1 enhances the interaction of Dscam1 with RPTP69D, stimulating Dscam1 dephosphorylation. Single-growth-cone imaging reveals that Slit/RPTP69D are not required for general branch initiation but instead promote the extension of specific axon collaterals. Hence, although regulation of intrinsic Dscam1-Dscam1 isoform interactions is essential for formation of all mechanosensory-axon branches, the local ligand-induced alterations of Dscam1 phosphorylation in distinct growth-cone compartments enable the spatial specificity of axon collateral formation.

Shin, D. H. and Hong, J. W. (2015). Midline enhancer activity of the short gastrulation shadow enhancer is characterized by three unusual features for cis-regulatory DNA. BMB Rep [Epub ahead of print]. PubMed ID: 26277983
The shadow enhancer of the short gastrulation (sog) gene directs its sequential expression in the neurogenic ectoderm and the ventral midline of the developing Drosophila embryo. This study characterized three unusual features of the shadow enhancer midline activity. First, the minimal regions for the two different enhancer activities exhibit high overlap within the shadow enhancer, meaning that one developmental enhancer possesses dual enhancer activities. Second, the midline enhancer activity relies on five Single-minded (Sim)-binding sites, two of which have not been found in any Sim target enhancer. Finally, two linked Dorsal (Dl)- and Zelda (Zld)-binding sites, critical for the neurogenic ectoderm enhancer activity, are also required for the midline enhancer activity. These results suggest that early activation by Dl and Zld may facilitate late activation via the noncanonical sites occupied by Sim. A model is described for Zld as a pioneer factor, and its role in midline enhancer activity is discussed.

Monday, September 28th

Asadzadeh, J., Neligan, N., Canabal-Alvear, J. J., Daly, A. C., Kramer, S. G. and Labrador, J. P. (2015). The Unc-5 Receptor Is Directly Regulated by Tinman in the Developing Drosophila Dorsal Vessel. PLoS One 10: e0137688. PubMed ID: 26356221
During early heart morphogenesis cardiac cells migrate in two bilateral opposing rows, meet at the dorsal midline and fuse to form a hollow tube known as the dorsal vessel (DV) in Drosophila. Guidance receptors are thought to mediate this evolutionarily conserved process. Whether the core transcription factors accomplish their function, at least in part, through direct or indirect transcriptional regulation of guidance receptors is currently unknown. This work demonstrates how Tinman (Tin), the Drosophila homolog of the Nkx-2.5 transcription factor, regulates the Unc-5 receptor during DV tube morphogenesis. Genetics, expression analysis with single cell mRNA resolution and enhancer-reporter assays in vitro or in vivo were used to demonstrate that Tin is required for Unc-5 receptor expression specifically in cardioblasts. Tin can bind to evolutionary conserved sites within an Unc-5 DV enhancer, and these sites were shown to be required for Tin-dependent transactivation both in vitro and in vivo (Asadzadeh, 2015).

Liu, J. and Ma, J. (2015). Modulation of temporal dynamics of gene transcription by activator potency in the Drosophila embryo. Development [Epub ahead of print]. PubMed ID: 26395487
The Drosophila embryo at the mid-blastula transition (MBT) experiences a concurrent receding of a first wave of zygotic transcription and surge of a massive second wave. It is not well understood how genes in the first wave become turned off transcriptionally and how their precise timing may impact embryonic development. This study perturbed the timing of the shutdown of Bicoid (Bcd)-dependent hunchback (hb) transcription in the embryo through the use of a Bcd mutant that has a heightened activating potency. A delayed shutdown increases specifically Bcd-activated hb levels that alter spatial characteristics of the patterning outcome and cause developmental defects. This study thus documents a specific participation of the maternal activator input strength in timing molecular events in precise accordance with the MBT morphological progression.

Ross, J., Kuzin, A., Brody, T. and Odenwald, W. F. (2015). cis-regulatory analysis of the Drosophila pdm locus reveals a diversity of neural enhancers. BMC Genomics 16: 700. PubMed ID: 26377945
Two of the regulators essential for neural diversity in Drosophila are the POU-domain TFs Nubbin and Pdm-2, encoded by adjacent genes collectively known as pdm. This study focussed on discovery and characterization of theis cis-regulatory DNA. Phylogenetic footprinting analysis of a 125 kb genomic region that spans the pdm locus identified 116 conserved sequence clusters. The screen revealed 77 unique enhancers positioned throughout the noncoding region of the pdm locus. Many of these activated neural-specific gene expression during different developmental stages and many drove expression in overlapping patterns. Sequence comparisons of functionally related enhancers that activate overlapping expression patterns revealed that they share conserved elements that can be predictive of enhancer behavior. The results of this analysis are catalogued in cisPatterns, an online database of the structure and function of these and other Drosophila enhancers. These studies reveal a diversity of modular enhancers that most likely regulate pdm gene expression during embryonic and adult development, highlighting a high level of temporal and spatial expression specificity. In addition, clusters of functionally related enhancers were discovered throughout the pdm locus. A subset of these enhancers share conserved elements including sequences that correspond to known TF DNA binding sites. Evidence is presented that although these two genes most likely arose from a duplication event, after this duplication their cis-regulatory DNA diverged at a rapid rate (Ross, 2015).

Eberle, A.B., Jordán-Pla, A., Gañez-Zapater, A., Hessle, V., Silberberg, G., von Euler, A., Silverstein, R.A. and Visa, N. (2015). An interaction between RRP6 and SU(VAR)3-9 targets RRP6 to heterochromatin and contributes to heterochromatin maintenance in Drosophila melanogaster. PLoS Genet 11: e1005523. PubMed ID: 26389589
RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. This study shows that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. It was shown that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network reveals physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrates that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, these results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin.

Sunday, September 27th

Wagner, A.E., et al. (2015). Epigallocatechin gallate affects glucose metabolism and increases fitness and lifespan in Drosophila melanogaster. Oncotarget [Epub ahead of print]. PubMed ID: 26375250
This study tested whether a standardized epigallocatechin-3-gallate (EGCG) rich green tea extract (comprising > 90% EGCG) affects fitness and lifespan as well as parameters of glucose metabolism and energy homeostasis in Drosophila. Application of the green tea extract resulted in a significant increase was detected in the mean lifespan and the 50% survival as well as improved fitnessd. These effects went along an increased expression of Spargel, the homolog of mammalian PGC1α, which has been reported to affect lifespan in flies. Intriguingly, in flies, treatment with the green tea extract decreases glucose concentrations, which are accompanied by an inhibition of α-amylase and α-glucosidase activity. Computational docking analysis proves the potential of EGCG to dock into the substrate binding pocket of α-amylase and to a greater extent into α-glucosidase. Furthermore, it was demonstrated that EGCG downregulates insulin-like peptide 5 and phosphoenolpyruvate carboxykinase, major regulators of glucose metabolism, as well as the Drosophila homolog of leptin, unpaired 2. The study proposes that a decrease in glucose metabolism in connection with an upregulated expression of Spargel contribute to the better fitness and the extended lifespan in EGCG-treated flies.

Huang, J.H. and Douglas, A.E. (2015). Consumption of dietary sugar by gut bacteria determines Drosophila lipid content. Biol Lett 11(9). PubMed ID: 26382071
Gut microorganisms are essential for the nutritional health of many animals, but the underlying mechanisms are poorly understood. This study investigated how lipid accumulation by adult Drosophila melanogaster is reduced in flies associated with the bacterium Acetobacter tropicalis which displays oral-faecal cycling between the gut and food. It was demonstrated that the lower lipid content of A. tropicalis-colonized flies relative to bacteria-free flies is linked with a parallel bacterial-mediated reduction in food glucose content; and can be accounted for quantitatively by the amount of glucose acquired by the flies, as determined from the feeding rate and assimilation efficiency of bacteria-free and A. tropicalis-colonized flies. The study recommends that nutritional studies on Drosophila include empirical quantification of food nutrient content, to account for likely microbial-mediated effects on diet composition. More broadly, the study demonstrates that selective consumption of dietary constituents by microorganisms can alter the nutritional balance of food and, thereby, influence the nutritional status of the animal host.

Chintapalli, V. R., Kato, A., Henderson, L., Hirata, T., Woods, D. J., Overend, G., Davies, S. A., Romero, M. F. and Dow, J. A. (2015). Transport proteins NHA1 and NHA2 are essential for survival, but have distinct transport modalities. Proc Natl Acad Sci U S A 112: 11720-11725. PubMed ID: 26324901
The cation/proton antiporter (CPA) family includes the well-known sodium/proton exchanger (NHE; SLC9A) family of Na(+)/H(+) exchangers, and the more recently discovered and less well understood CPA2s (SLC9B), found widely in living organisms. In Drosophila, as in humans, they are represented by two genes, Nha1 (Slc9b1) and Nha2 (Slc9b2), which are enriched and functionally significant in renal tubules. The importance of their role in organismal survival has not been investigated in animals, however. This study shows that single RNAi knockdowns of either Nha1 or Nha2 reduce survival and in combination are lethal. Knockdown of either gene alone results in up-regulation of the other, suggesting functional complementation of the two genes. Under salt stress, knockdown of either gene decreases survival, demonstrating a key role for the CPA2 family in ion homeostasis. This is specific to Na(+) stress; survival on K(+) intoxication is not affected by sodium/hydrogen antiporter (NHA) knockdown. A direct functional assay in Xenopus oocytes shows that Nha2 acts as a Na(+)/H(+) exchanger. In contrast, Nha1 expressed in Xenopus oocytes shows strong Cl(-) conductance and acts as a H(+)-Cl(-) cotransporter. The activity of Nha1 is inhibited by chloride-binding competitors. Salt stress induces a massive up-regulation of NHA gene expression not in the major osmoregulatory tissues of the alimentary canal, but in the crop, cuticle, and associated tissues. Thus, it is necessary to revise the classical view of the coordination of different tissues in the coordination of the response to osmoregulatory stress.

Galikova, M., Diesner, M., Klepsatel, P., Hehlert, P., Xu, Y., Bickmeyer, I., Predel, R. and Kuhnlein, R. P. (2015). Energy homeostasis control in Drosophila adipokinetic hormone mutants. Genetics [Epub ahead of print]. PubMed ID: 26275422
Maintenance of biological functions under negative energy balance depends on mobilization of storage lipids and carbohydrates in animals. In mammals, glucagon and glucocorticoid signaling mobilizes energy reserves, whereas Adipokinetic hormones (AKHs) play a homologous role in insects. Numerous studies based in AKH injections and correlative studies in a broad range of insect species established the view that AKH acts as master regulator of energy mobilization during development, reproduction, and stress. In contrast to AKH, the second peptide, which is processed from the Akh encoded prohormone - termed Adipokinetic hormone precursor related peptide (APRP) - is functionally orphan. APRP is discussed as ecdysiotropic hormone or as scaffold peptide during AKH prohormone processing. However, as in the case of AKH, final evidence for APRP functions requires genetic mutant analysis. This study employed CRISPR/Cas9-mediated genome engineering to create AKH and AKH plus APRP-specific mutants in the model insect Drosophila melanogaster. Lack of APRP did not affect any of the tested steroid-dependent processes. Similarly, Drosophila AKH signaling is dispensable for ontogenesis, locomotion, oogenesis, and homeostasis of lipid or carbohydrate storage until up to the end of metamorphosis. During adulthood, however, AKH regulates body fat content and the hemolymph sugar level as well as nutritional and oxidative stress responses. Finally, evidence is provided for a negative auto-regulatory loop, in Akh gene regulation.

Saturday, September 26th

Helmfors, L., Boman, A., Civitelli, L., Nath, S., Sandin, L., Janefjord, C., McCann, H., Zetterberg, H., Blennow, K., Halliday, G., Brorsson, A. C. and Kagedal, K. (2015). Protective properties of lysozyme on beta-amyloid pathology: implications for Alzheimer disease. Neurobiol Dis 83: 122-133. PubMed ID: 26334479
The hallmarks of Alzheimer disease are amyloid-beta plaques and neurofibrillary tangles accompanied by signs of neuroinflammation. Lysozyme is a major player in the innate immune system and has recently been shown to prevent the aggregation of amyloid-beta1-40in vitro. This study found that patients with Alzheimer disease have increased lysozyme levels in the cerebrospinal fluid and lysozyme co-localized with amyloid-β in plaques. In Drosophila neuronal co-expression of lysozyme and amyloid-β1-42 (see Drosophila Appl) reduced the formation of soluble and insoluble amyloid-β species, prolonged survival and improved the activity of amyloid-β1-42 transgenic flies. This suggests that lysozyme levels rise in Alzheimer disease as a compensatory response to amyloid-β increases and aggregation. In support of this, in vitro aggregation assays revealed that lysozyme associates with amyloid-β1-42 and alters its aggregation pathway to counteract the formation of toxic amyloid-β species. Overall, these studies establish a protective role for lysozyme against amyloid-β associated toxicities and identify increased lysozyme in patients with Alzheimer disease. Therefore, lysozyme has potential as a new biomarker as well as a therapeutic target for Alzheimer disease.

Zarndt, R., Piloto, S., Powell, F.L., Haddad, G.G., Bodmer, R. and Ocorr, K. (2015). Cardiac responses to hypoxia and reoxygenation in Drosophila. Am J Physiol Regul Integr Comp Physiol [Epub ahead of print]. PubMed ID: 26377557
An adequate supply of oxygen is important for the survival of all tissues, but is especially critical for tissues with high energy demands such as the heart. This study used the genetic model system Drosophila to investigate cardiac responses to acute (30 minutes), sustained (18 hours), and chronic (3 weeks) hypoxia with reoxygenation. Whereas hearts from wild type flies recover quickly after acute hypoxia, exposure to sustained or chronic hypoxia significantly compromises heart function upon reoxygenation. Hearts from flies with mutations in sima, Drosophila homolog of the Hypoxia Inducible Factor alpha subunit (HIFα), exhibit exaggerated reductions in cardiac output in response to hypoxia. Heart function in hypoxia-selected flies, selected over many generations for survival in a low oxygen environment, reveals reduced cardiac output in terms of decreased heart rate and fractional shortening compared to their normoxia controls. Hypoxia-selected flies also have smaller hearts, myofibrillar disorganization and increased extracellular collagen deposition, consistent with the observed reductions in contractility. This study indicates that longer duration hypoxic insults exert deleterious effects on heart function that are mediated in part by sima and advances Drosophila models for the genetic analysis of cardiac-specific responses to hypoxia and reoxygenation.

Suzuki, M., Fujikake, N., Takeuchi, T., Kohyama-Koganeya, A., Nakajima, K., Hirabayashi, Y., Wada, K. and Nagai, Y. (2015). Glucocerebrosidase deficiency accelerates the accumulation of proteinase K-resistant α-synuclein and aggravates neurodegeneration in a Drosophila model of Parkinson's disease. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26362253
Alpha-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Recent multicenter genetic studies have revealed that mutations in the glucocerebrosidase 1 (GBA1) gene, which are responsible for Gaucher's disease, are strong risk factors for PD and DLB. However, the mechanistic link between the functional loss of glucocerebrosidase (GCase) and the toxicity of αSyn in vivo is not fully understood. This study employed Drosophila models to examine the effect of GCase deficiency on the neurotoxicity of αSyn and its molecular mechanism. Behavioral and histological analyses showed that knockdown of the Drosophila homologue of GBA1 (dGBA1) exacerbates the locomotor dysfunction, loss of dopaminergic neurons, and retinal degeneration of αSyn-expressing flies. This phenotypic aggravation was associated with the accumulation of proteinase K (PK)-resistant αSyn, rather than with changes in the total amount of αSyn, raising the possibility that glucosylceramide (GlcCer), a substrate of GCase, accelerates the misfolding of αSyn. Indeed, in vitro experiments revealed that GlcCer directly promotes the conversion of recombinant αSyn into the PK-resistant form, representing a toxic conformational change. Similarly to dGBA1 knockdown, knockdown of the Drosophila homologue of β-galactosidase (β-Gal) also aggravated locomotor dysfunction of the αSyn flies, and its substrate GM1 ganglioside accelerated the formation of PK-resistant αSyn. These findings suggest that the functional loss of GCase or β-Gal promotes the toxic conversion of αSyn via aberrant interactions between αSyn and their substrate glycolipids, leading to the aggravation of αSyn-mediated neurodegeneration.

Kong, Y., Liang, X., Liu, L., Zhang, D., Wan, C., Gan, Z. and Yuan, L. (2015). High throughput sequencing identifies microRNAs mediating α-synuclein toxicity by targeting neuroactive-ligand receptor interaction pathway in early stage of Drosophila Parkinson's disease model. PLoS One 10: e0137432. PubMed ID: 26361355
Parkinson's disease (PD) is a prevalent neurodegenerative disorder with pathological features including death of dopaminergic neurons in the substantia nigra and intraneuronal accumulations of Lewy bodies. As the main component of Lewy bodies, α-synuclein is implicated in PD pathogenesis by aggregation into insoluble filaments. However, the detailed mechanisms underlying α-synuclein induced neurotoxicity in PD are still elusive.├čBy using Drosophila PD model expressing α-synuclein A30P, this study examined brain miRNA expression with high-throughput small RNA sequencing technology. It was found that five miRNAs (dme-miR-133-3p, dme-miR-137-3p, dme-miR-13b-3p, dme-miR-932-5p, dme-miR-1008-5p) were upregulated in PD flies. Among them, miR-13b, miR-133, miR-137 are brain enriched and highly conserved from Drosophila to humans. KEGG pathway analysis using DIANA miR-Path demonstrated that neuroactive-ligand receptor interaction pathway is most likely affected by these miRNAs. Interestingly, miR-137 has been predicted to regulate most of the identified targets in this pathway, including dopamine receptor (DopR, D2R), γ-aminobutyric acid (GABA) receptor (GABA-B-R1, GABA-B-R3) and N-methyl-D-aspartate (NMDA) receptor (Nmdar2). The validation experiments showed that the expression of miR-137 and its targets is negatively correlated in PD flies. Further experiments using luciferase reporter assay confirmed that miR-137 could act on specific sites in 3' UTR region of D2R, Nmdar2 and GABA-B-R3, which downregulated significantly in PD flies. Collectively, these findings indicate that α-synuclein could induce the dysregulation of miRNAs, which target neuroactive ligand-receptor interaction pathway in vivo.

Friday, September 25th

Collinet, C., Rauzi, M., Lenne, P.F. and Lecuit, T. (2015). Local and tissue-scale forces drive oriented junction growth during tissue extension. Nat Cell Biol [Epub ahead of print]. PubMed ID: 26389664
Convergence-extension is a widespread morphogenetic process driven by polarized cell intercalation. In the Drosophila germ band, epithelial intercalation comprises loss of junctions between anterior-posterior neighbours followed by growth of new junctions between dorsal-ventral neighbours. Much is known about how active stresses drive polarized junction shrinkage. However, it is unclear how tissue convergence-extension emerges from local junction remodelling and what the specific role, if any, of junction growth is. This study reports that tissue convergence and extension correlate mostly with new junction growth. Simulations and in vivo mechanical perturbations reveal that junction growth is due to local polarized stresses driven by medial actomyosin contractions. Moreover, it was found that tissue-scale pulling forces at the boundary with the invaginating posterior midgut actively participate in tissue extension by orienting junction growth. Thus, tissue extension is akin to a polarized fluid flow that requires parallel and concerted local and tissue-scale forces to drive junction growth and cell-cell displacement.

Schweizer, N., Pawar, N., Weiss, M. and Maiato, H. (2015). An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region. J Cell Biol 210: 695-704. PubMed ID: 26304726
The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. This paper describes evidence for a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, this study demonstrates that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous 'spindle envelope' confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function.

Smyth, J. T., Schoborg, T. A., Bergman, Z. J., Riggs, B. and Rusan, N. M. (2015). Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules. Open Biol 5. PubMed ID: 26289801
Mechanisms that regulate partitioning of the endoplasmic reticulum (ER) during cell division are largely unknown. Previous studies have mostly addressed ER partitioning in cultured cells, which may not recapitulate physiological processes that are critical in developing, intact tissues. This has been addressed by analysing ER partitioning in asymmetrically dividing stem cells, in which precise segregation of cellular components is essential for proper development and tissue architecture. In Drosophila neural stem cells, called neuroblasts, the ER asymmetrically partitioned to centrosomes early in mitosis. This correlated closely with the asymmetric nucleation of astral microtubules (MTs) by centrosomes, suggesting that astral MT association may be required for ER partitioning by centrosomes. Consistent with this, the ER also associated with astral MTs in meiotic Drosophila spermatocytes and during syncytial embryonic divisions. Disruption of centrosomes in each of these cell types led to improper ER partitioning, demonstrating the critical role for centrosomes and associated astral MTs in this process. Importantly, this study showed that the ER also associated with astral MTs in cultured human cells, suggesting that this centrosome/astral MT-based partitioning mechanism is conserved across animal species.

Winkler, F., Gummalla, M., Kunneke, L., Lv, Z., Zippelius, A., Aspelmeier, T. and Grosshans, J. (2015). Fluctuation analysis of centrosomes reveals a cortical function of Kinesin-1. Biophys J 109: 856-868. PubMed ID: 26331244
The actin and microtubule networks form the dynamic cytoskeleton. Network dynamics is driven by molecular motors applying force onto the networks and the interactions between the networks. This study assayed the dynamics of centrosomes in the scale of seconds as a proxy for the movement of microtubule asters. This assay was designed to detect the role of specific motors and of network interaction. During interphase of syncytial embryos of Drosophila, cortical actin and the microtubule network depend on each other. Centrosomes induce cortical actin to form caps, whereas F-actin anchors microtubules to the cortex. In addition, lateral interactions between microtubule asters are assumed to be important for regular spatial organization of the syncytial embryo. This study recorded the movement of centrosomes at 1 Hz and analyzed their fluctuations. F-actin was found to be required for directional movements during initial centrosome pair separation. By analysis of mutant and drug-injected embryos, it was found that the fluctuations were suppressed by both cortical actin and microtubules. Surprisingly, the microtubule motor Kinesin-1 also suppressed fluctuations to a similar degree as F-actin. Kinesin-1 may mediate linkage of the microtubule (+)-ends to the actin cortex. Consistent with this model is that finding that Kinesin-1-GFP accumulates at the cortical actin caps.

Thursday, September 24th

Polak, G. L., Pasqualino, A., Docherty, J. E., Beck, S. J. and DiAngelo, J. R. (2015). The regulation of muscle structure and metabolism by Mio/dChREBP in Drosophila. PLoS One 10: e0136504. PubMed ID: 26305467
All cells require energy to perform their specialized functions. Muscle is particularly sensitive to the availability of nutrients due to the high-energy requirement for muscle contraction. Therefore the ability of muscle cells to obtain, store and utilize energy is essential for the function of these cells. Mio, the Drosophila homolog of carbohydrate response element binding protein (ChREBP), has recently been identified as a nutrient responsive transcription factor important for triglyceride storage in the fly fat body. However, the function of Mio in muscle is unknown. This study, characterized the role of Mio in controlling muscle function and metabolism. Decreasing Mio levels using RNAi specifically in muscle results in increased thorax glycogen storage. Adult Mio-RNAi flies also have a flight defect due to altered myofibril shape and size in the indirect flight muscles as shown by electron microscopy. Myofibril size is also decreased in flies just before emerging from their pupal cases, suggesting a role for Mio in myofibril development. Together, these data indicate a novel role for Mio in controlling muscle structure and metabolism and may provide a molecular link between nutrient availability and muscle function.

Barrios, N., Gonzalez-Perez, E., Hernandez, R. and Campuzano, S. (2015). The homeodomain Iroquois proteins control cell cycle progression and regulate the size of developmental fields. PLoS Genet 11: e1005463. PubMed ID: 26305360
During development, proper differentiation and final organ size rely on the control of territorial specification and cell proliferation. Although many regulators of these processes have been identified, how both are coordinated remains largely unknown. The homeodomain Iroquois/Irx proteins (see Caupolican) play a key, evolutionarily conserved, role in territorial specification. This study shows that in the imaginal discs, reduced function of Iroquois genes promotes cell proliferation by accelerating the G1 to S transition. Conversely, their increased expression causes cell-cycle arrest, down-regulating the activity of the Cyclin E/Cdk2 complex. This study demonstrates that physical interaction of the Iroquois protein Caupolican with Cyclin E-containing protein complexes, through its IRO box and Cyclin-binding domains, underlies its activity in cell-cycle control. Thus, Drosophila Iroquois proteins are able to regulate cell-autonomously the growth of the territories they specify. Moreover, the results provide a molecular mechanism for a role of Iroquois/Irx genes as tumour suppressors.

Bolstad, G.H., Cassara, J.A., Márquez, E., Hansen, T.F., van der Linde, K., Houle, D. and Pélabon, C. (2015). Complex constraints on allometry revealed by artificial selection on the wing of Drosophila melanogaster. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26371319
Precise exponential scaling with size is a fundamental aspect of phenotypic variation. These allometric power laws are often invariant across taxa and have long been hypothesized to reflect developmental constraints. This study tests this hypothesis by investigating the evolutionary potential of an allometric scaling relationship in drosophilid wing shape that is nearly invariant across 111 species separated by at least 50 million years of evolution. In only 26 generations of artificial selection in a population of Drosophila melanogaster, the allometric slope could be driven to the outer range of those found among the 111 sampled species. This response was rapidly lost when selection was suspended. Only a small proportion of this reversal could be explained by breakup of linkage disequilibrium, and direct selection on wing shape is also unlikely to explain the reversal, because the more divergent wing shapes produced by selection on the allometric intercept did not revert. The study hypothesizes that the reversal is instead caused by internal selection arising from pleiotropic links to unknown traits. These results also suggest that the observed selection response in the allometric slope is due to a component expressed late in larval development and that variation in earlier development did not respond to selection. Together, these results are consistent with a role for pleiotropic constraints in explaining the remarkable evolutionary stability of allometric scaling.

Ivy, J. R., Drechsler, M., Catterson, J. H., Bodmer, R., Ocorr, K., Paululat, A. and Hartley, P. S. (2015). Klf15 is critical for the development and differentiation of Drosophila nephrocytes. PLoS One 10: e0134620. PubMed ID: 26301956
Insect nephrocytes are highly endocytic scavenger cells that represent the only invertebrate model for the study of human kidney podocytes. Despite their importance, nephrocyte development is largely uncharacterised. This work tested whether the insect ortholog of mammalian Kidney Kruppel-Like Factor (Klf15), a transcription factor required for mammalian podocyte differentiation, was required for insect nephrocyte development. Expression of Drosophila Klf15 (dKlf15, previously known as Bteb2) was found to be restricted to the only two nephrocyte populations in Drosophila, the garland cells and pericardial nephrocytes. Loss of dKlf15 function led to attrition of both nephrocyte populations and sensitised larvae to the xenotoxin silver nitrate. Although pericardial nephrocytes in dKlf15 loss of function mutants were specified during embryogenesis, they failed to express the slit diaphragm gene sticks and stones and did not form slit diaphragms. Conditional silencing of dKlf15 in adults led to reduced surface expression of the endocytic receptor Amnionless and loss of in vivo scavenger function. Over-expression of dKlf15 increased nephrocyte numbers and rescued age-dependent decline in nephrocyte function. The data place dKlf15 upstream of sns and Amnionless in a nephrocyte-restricted differentiation pathway and suggest dKlf15 expression is both necessary and sufficient to sustain nephrocyte differentiation. These findings explain the physiological relevance of dKlf15 in Drosophila and imply that the role of KLF15 in human podocytes is evolutionarily conserved.

Wednesday, September 23rd

Pooryasin, A. and Fiala, A. (2015). Identified serotonin-releasing neurons induce behavioral quiescence and suppress mating in Drosophila. J Neurosci 35: 12792-12812. PubMed ID: 26377467
Animals show different levels of activity that are reflected in sensory responsiveness and endogenously generated behaviors. Biogenic amines have been determined to be causal factors for these states of arousal. It is well established that, in Drosophila, dopamine and octopamine promote increased arousal. However, little is known about factors that regulate arousal negatively and induce states of quiescence. Moreover, it remains unclear whether global, diffuse modulatory systems comprehensively affecting brain activity determine general states of arousal. Alternatively, individual aminergic neurons might selectively modulate the animals' activity in a distinct behavioral context. This study shows that artificially activating large populations of serotonin-releasing neurons induces behavioral quiescence and inhibits feeding and mating. The study systematically narrows down a role of serotonin in inhibiting endogenously generated locomotor activity to neurons located in the posterior medial protocerebrum. Neurons of this cell cluster were identified that suppress mating, but not feeding behavior. These results suggest that serotonin does not uniformly act as global, negative modulator of general arousal. Rather, distinct serotoninergic neurons can act as inhibitory modulators of specific behaviors.

Flourakis, M., Kula-Eversole, E., Hutchison, A. L., Han, T. H., Aranda, K., Moose, D. L., White, K. P., Dinner, A. R., Lear, B. C., Ren, D., Diekman, C. O., Raman, I. M. and Allada, R. (2015). A conserved bicycle model for circadian clock control of membrane excitability. Cell 162: 836-848. PubMed ID: 26276633
Circadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. This study found that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel Narrow abdomen depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1 (CG10420), linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, this study reveals an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake.

Clowney, E. J., Iguchi, S., Bussell, J. J., Scheer, E. and Ruta, V. (2015). Multimodal chemosensory circuits controlling male courtship in Drosophila. Neuron 87: 1036-1049. PubMed ID: 26279475
Throughout the animal kingdom, internal states generate long-lasting and self-perpetuating chains of behavior. In Drosophila, males instinctively pursue females with a lengthy and elaborate courtship ritual triggered by activation of sexually dimorphic P1 interneurons. P1 interneurons, located in the dorsal posterior brain near the mushroom body, is composed of 20 interneurons, each of which has a primary transversal neurite with extensive ramifications in the bilateral protocerebrum. P1 is fated to die in females through the action of a feminizing protein, DsxF. A masculinizing protein Fru is required in the male brain for correct positioning of the terminals of P1 neurites. Gustatory pheromones are thought to activate P1 neurons but the circuit mechanisms that dictate their sensory responses to gate entry into courtship remain unknown. This study used circuit mapping and in vivo functional imaging techniques to trace gustatory and olfactory pheromone circuits to their point of convergence onto P1 neurons and reveals how their combined input underlies selective tuning to appropriate sexual partners. Inhibition, even in response to courtship-promoting pheromones, was identified as a key circuit element that tunes and tempers P1 neuron activity. The results suggest a circuit mechanism in which balanced excitation and inhibition underlie discrimination of prospective mates and stringently regulate the transition to courtship in Drosophila.

Kadas, D., Ryglewski, S. and Duch, C. (2015). Transient BK outward current enhances motoneurone firing rates during Drosophila larval locomotion. J Physiol [Epub ahead of print]. PubMed ID: 26332699
A large number of voltage gated ion channels, their interactions with accessory subunits, and their posttranscriptional modifications generate an immense functional diversity of neurones. Therefore, a key challenge is to understand the genetic basis and precise function of specific ionic conductances for neuronal firing properties in the context of behavior. This study identifies slowpoke (slo) as exclusively mediating fast activating, fast inactivating BK current (ICF) in larval Drosophila crawling motoneurones. Combining in vivo patch clamp recordings during larval crawling with pharmacology and targeted genetic manipulations reveals that ICF acts specifically in motoneurones to sculpt their firing patterns in response to a given input from the central pattern generating (CPG) networks. First, ICF curtails motoneurone postsynaptic depolarizations during rhythmical CPG drive. Second, ICF is activated during the rising phase of the action potential and mediates a fast afterhyperpolarization. Consequently, ICF is required for maximal intraburst firing rates during locomotion, likely by allowing recovery from inactivation of fast sodium channels and decreased potassium channel activation. This contrasts the common view that outward conductances oppose excitability, but is in accord with reports on transient BK (see Drosophila Slowpoke) as well as Kv3 channel (see Drosophila Shawl) function in multiple types of vertebrate neurones. Therefore, this finding that ICF enhances firing rates specifically during bursting patterns relevant to behavior is likely of relevance to all brains.

Garlapow, M.E., Huang, W., Yarboro, M.T., Peterson, K.R. and Mackay, T.F. (2015). Quantitative genetics of food intake in Drosophila melanogaster. PLoS One 10: e0138129. PubMed ID: 26375667
Food intake is an essential animal activity, regulated by neural circuits that motivate food localization, evaluate nutritional content and acceptance or rejection responses through the gustatory system, and regulate neuroendocrine feedback loops that maintain energy homeostasis. Excess food consumption in people is associated with obesity and metabolic and cardiovascular disorders. However, little is known about the genetic basis of natural variation in food consumption. To gain insights in evolutionarily conserved genetic principles that regulate food intake, this study took advantage of a model system, Drosophila melanogaster, in which food intake, environmental conditions and genetic background can be controlled precisely. The study quantified variation in food intake among 182 inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel (DGRP). A significant genetic variation in the mean and within-line environmental variance of food consumption was found and sexual dimorphism and genetic variation in sexual dimorphism for both food intake traits (mean and variance) was observed. Genome wide association (GWA) analyses was performed for mean food intake and environmental variance of food intake (using the coefficient of environmental variation, CVE, as the metric for environmental variance) and molecular polymorphisms associated with both traits were identified. Validation experiments using RNAi-knockdown confirms 24 of 31 (77%) candidate genes affecting food intake and/or variance of food intake, and a test cross between selected DGRP lines confirms a SNP affecting mean food intake identified in the GWA analysis. The majority of the validated candidate genes are novel with respect to feeding behavior, and many have mammalian orthologs implicated in metabolic diseases.

Itakura, Y., Kohsaka, H., Ohyama, T., Zlatic, M., Pulver, S. R. and Nose, A. (2015). Identification of inhibitory premotor interneurons activated at a late phase in a motor cycle during Drosophila larval locomotion. PLoS One 10: e0136660. PubMed ID: 26335437
Rhythmic motor patterns underlying many types of locomotion are thought to be produced by central pattern generators (CPGs). This study used the motor circuitry underlying crawling in larval Drosophila as a model to try to understand how segmentally coordinated rhythmic motor patterns are generated. Whereas muscles, motoneurons and sensory neurons have been well investigated in this system, far less is known about the identities and function of interneurons. A recent study identified a class of glutamatergic premotor interneurons, PMSIs (period-positive median segmental interneurons), that regulate the speed of locomotion. This study reports on the identification of a distinct class of glutamatergic premotor interneurons called Glutamatergic Ventro-Lateral Interneurons (GVLIs). Calcium imaging was used to search for interneurons that show rhythmic activity, and GVLIs were identified as interneurons showing wave-like activity during peristalsis. Paired GVLIs were present in each abdominal segment A1-A7 and locally extended an axon towards a dorsal neuropile region, where they formed GRASP-positive putative synaptic contacts with motoneurons. The interneurons expressed vesicular glutamate transporter (vGluT) and thus likely secrete glutamate, a neurotransmitter known to inhibit motoneurons. These anatomical results suggest that GVLIs are premotor interneurons that locally inhibit motoneurons in the same segment. Consistent with this, optogenetic activation of GVLIs with the red-shifted channelrhodopsin, CsChrimson ceased ongoing peristalsis in crawling larvae. Simultaneous calcium imaging of the activity of GVLIs and motoneurons showed that GVLIs' wave-like activity lagged behind that of motoneurons by several segments. Thus, GVLIs are activated when the front of a forward motor wave reaches the second or third anterior segment. It is proposed that GVLIs are part of the feedback inhibition system that terminates motor activity once the front of the motor wave proceeds to anterior segments.

Tuesday, September 22nd

Blagodatski, A., Sergeev, A., Kryuchkov, M., Lopatina, Y. and Katanaev, V.L. (2015). Diverse set of Turing nanopatterns coat corneae across insect lineages. Proc Natl Acad Sci U S A 112: 10750-10755. PubMed ID: 26307762
Nipple-like nanostructures covering the corneal surfaces of moths, butterflies, and Drosophila have been studied by electron and atomic force microscopy, and their antireflective properties have been described. In contrast, corneal nanostructures of the majority of other insect orders have either been unexamined or examined by methods that did not allow precise morphological characterization. This study provides a comprehensive analysis of corneal surfaces in 23 insect orders, revealing a rich diversity of insect corneal nanocoatings. These nanocoatings are categorized into four major morphological patterns and various transitions between them, many, never described before. Remarkably, this unexpectedly diverse range of the corneal nanostructures replicates the complete set of Turing patterns, thus likely being a result of processes similar to those modeled by Alan Turing in his famous reaction-diffusion system. These findings reveal a beautiful diversity of insect corneal nanostructures and shed light on their molecular origin and evolutionary diversification. They may also be the first-ever biological example of Turing nanopatterns.

Ma, H. and O'Farrell, P. H. (2015). Selections that isolate recombinant mitochondrial genomes in animals. Elife 4 [Epub ahead of print]. PubMed ID: 26237110
Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. This study designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germ line and revealed somatic recombination. When the recombination partner was a diverged D. melanogaster genome or a genome from a different species such as D. yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed mapping of a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome.

Jackson, S., Nielsen, D. M. and Singh, N. D. (2015). Increased exposure to acute thermal stress is associated with a non-linear increase in recombination frequency and an independent linear decrease in fitness in Drosophila. BMC Evol Biol 15: 175. PubMed ID: 26310872
Meiotic recombination rate has long been known to be phenotypically plastic. How plastic recombination evolves and is maintained remains controversial; though a leading model for the evolution of plastic recombination rests on the tenet that organismal fitness and recombination frequency are negatively correlated. Motivated by the mounting evidence that meiotic recombination frequencies increase in response to stress, this study tested for a negative correlation between fitness and recombination frequency. Specifically, the fitness-associated recombination model (FAR) predicts that if stress increases meiotic recombination frequency, then increasing exposure to stressful conditions will yield an increasing magnitude of the recombinational response, while concomitantly decreasing fitness. This study used heat shock as a stressor to test this prediction in Drosophila melanogaster. Increased exposure to heat shock conditions was associated with a non-linear increase in meiotic recombination frequency. An independent effect of heat shock on organismal fitness was found, with fitness decreasing with increased duration of thermal stress. These results thus support the foundation of the FAR model for the evolution of plastic recombination. The data also suggest that modulating recombination frequency is one mechanism by which organisms can rapidly respond to environmental cues and confer increased adaptive potential to their offspring.

Flores, H. A., Bubnell, J. E., Aquadro, C. F. and Barbash, D. A. (2015). The Drosophila bag of marbles gene interacts genetically with Wolbachia and shows female-specific effects of divergence. PLoS Genet 11: e1005453. PubMed ID: 26291077
Many reproductive proteins from diverse taxa evolve rapidly and adaptively. These proteins are typically involved in late stages of reproduction such as sperm development and fertilization, and are more often functional in males than females. Surprisingly, many germline stem cell (GSC) regulatory genes, which are essential for the earliest stages of reproduction, also evolve adaptively in Drosophila. One example is the bag of marbles (bam) gene, which is required for GSC differentiation and germline cyst development in females and for regulating mitotic divisions and entry to spermatocyte differentiation in males. This study shows that the extensive divergence of bam between Drosophila melanogaster and D. simulans affects bam function in females but has no apparent effect in males. It was further found that infection with Wolbachia pipientis, an endosymbiotic bacterium that can affect host reproduction through various mechanisms, partially suppresses female sterility caused by bam mutations in D. melanogaster and interacts differentially with bam orthologs from D. melanogaster and D. simulans. It is proposed that the adaptive evolution of bam has been driven at least in part by the long-term interactions between Drosophila species and Wolbachia. More generally, it is suggested that microbial infections of the germline may explain the unexpected pattern of evolution of several GSC regulatory genes.

Choi, J. Y., Bubnell, J. E. and Aquadro, C. F. (2015). Population genomics of infectious and integrated Wolbachia pipientis genomes in Drosophila ananassae. Genome Biol Evol 7: 2362-2382. PubMed ID: 26254486
Coevolution between Drosophila and its endosymbiont Wolbachia pipientis has many intriguing aspects. For example, Drosophila ananassae hosts two forms of W. pipientis genomes: One being the infectious bacterial genome and the other integrated into the host nuclear genome. This study characterized the infectious and integrated genomes of W. pipientis infecting D. ananassae (wAna), by genome sequencing 15 strains of D. ananassae that have either the infectious or integrated wAna genomes. Results indicate evolutionarily stable maternal transmission for the infectious wAna genome suggesting a relatively long-term coevolution with its host. In contrast, the integrated wAna genome showed pseudogene-like characteristics accumulating many variants that are predicted to have deleterious effects if present in an infectious bacterial genome. Phylogenomic analysis of sequence variation together with genotyping by polymerase chain reaction of large structural variations indicated several wAna variants among the eight infectious wAna genomes. In contrast, only a single wAna variant was found among the seven integrated wAna genomes examined in lines from Africa, south Asia, and south Pacific islands suggesting that the integration occurred once from a single infectious wAna genome and then spread geographically. Further analysis revealed that for all D. ananassae examined with the integrated wAna genomes, the majority of the integrated wAna genomic regions is represented in at least two copies suggesting a double integration or single integration followed by an integrated genome duplication. The possible evolutionary mechanism underlying the widespread geographical presence of the duplicate integration of the wAna genome is an intriguing question remaining to be answered.

Hoang, D., Kopp, A. and Chandler, J. A. (2015). Interactions between Drosophila and its natural yeast symbionts - Is Saccharomyces cerevisiae a good model for studying the fly-yeast relationship?. PeerJ 3: e1116. PubMed ID: 26336636
Yeasts play an important role in the biology of the fruit fly, Drosophila melanogaster. This study explored the D. melanogaster-yeast relationship using five different strains of yeast that were isolated from wild Drosophila populations. Ingested live yeasts have variable persistence in the D. melanogaster gastrointestinal tract. For example, Hanseniaspora occidentalis persists relative to S. cerevisiae, while Brettanomyces naardenensis is removed. Despite these differences in persistence relative to S. cerevisiae, it was found that all yeasts decrease in total abundance over time. Reactive oxygen species (ROS) are an important component of the D. melanogaster anti-microbial response and can inhibit S. cerevisiae growth in the intestine. To determine if sensitivity to ROS explains the differences in yeast persistence, yeast growth was measured in the presence and absence of hydrogen peroxide. B. naardenesis is completely inhibited by hydrogen peroxide, while H. occidentalis is not, which is consistent with yeast sensitivity to ROS affecting persistence within the D. melanogaster gastrointestinal tract. The feeding preference of D. melanogaster was compared when given the choice between a naturally associated yeast and S. cerevisiae. No correlation was found between preferred yeasts and those that persist in the intestine. Notably, in no instances is S. cerevisiae preferred over the naturally associated strains. Overall, these results show that D. melanogaster-yeast interactions are more complex than might be revealed in experiments that use only S. cerevisiae. It is proposed that future research utilize other yeasts, and especially those that are naturally associated with Drosophila, to more fully understand the role of yeasts in Drosophila biology.

Rundle, H. D. and Dyer, K. A. (2015). Reproductive character displacement of female mate preferences for male cuticular hydrocarbons in Drosophila subquinaria. Evolution [Epub ahead of print]. PubMed ID: 26299584
Several lines of evidence implicate sexual isolation in both initiating and completing the speciation process. This study examined geographic variation in female mate preferences for male sexual displays in the fly Drosophila subquinaria. Female D. subquinaria that are sympatric with its sister species D. recens discriminate strongly against both D. recens and allopatric conspecific males, whereas females from allopatric populations do not. Furthermore, female mate preferences target at least in part a suite of cuticular hydrocarbons (CHCs) in males and geographic variation in CHCs mirrors the pattern of mate discrimination. This study quantified female mate preferences for male CHCs from populations that span the geographic range of D. subquinaria. The direction of linear sexual selection was found to vary significantly between populations that are sympatric versus allopatric with D. recens, in a pattern of reproductive character displacement. Differences in preference partially align with existing differences in CHCs and patterns of sexual isolation, although discrepancies remain that suggest the involvement of additional traits and/or more complex, nonlinear preference functions.

Najarro, M. A., Sumethasorn, M., Lamoureux, A. and Turner, T. L. (2015). Choosing mates based on the diet of your ancestors: replication of non-genetic assortative mating in Drosophila melanogaster. PeerJ 3: e1173. PubMed ID: 26339551
Assortative mating has been a focus of considerable research because of its potential to influence biodiversity at many scales. Sharon (2010) discovered that an inbred strain of Drosophila melanogaster mated assortatively based on the diet of previous generations, leading to initial reproductive isolation without genetic evolution. This behavior was reproduced by manipulating the microbiome independently of the diet, pointing to extracellular bacterial symbionts as the assortative mating cue. To further investigate the biological significance of this result, this study attempted to reproduce this phenomenon in an independent laboratory using different genotypes and additional mating assays. Supporting the previous result, it was found that a different inbred strain also mated assortatively based on the diets of previous generations. However, generation of assortative mating in an outbred strain from North Carolina was unsuccessful. The results support the potential for non-genetic mechanisms to influence reproductive isolation, but additional work is needed to investigate the importance of this mechanism in natural populations of Drosophila.

Monday, September 21st

Zhang, W., Cheng, L.E., Kittelmann, M., Li, J., Petkovic, M., Cheng, T., Jin, P., Guo, Z., Göpfert, M.C., Jan, L.Y. and Jan, Y.N. (2015). Ankyrin repeats convey force to gate the NOMPC mechanotransduction channel. Cell 162: 13903. PubMed ID: 26359990
How metazoan mechanotransduction channels sense mechanical stimuli is not well understood. The NOMPC channel in the transient receptor potential (TRP) family, a mechanotransduction channel for Drosophila touch sensation and hearing, contains 29 Ankyrin repeats (ARs) that associate with microtubules. These ARs have been postulated to act as a tether that conveys force to the channel. This study reports that these N-terminal ARs form a cytoplasmic domain essential for NOMPC mechanogating in vitro, mechanosensitivity of touch receptor neurons in vivo, and touch-induced behaviors of Drosophila larvae. Duplicating the ARs elongates the filaments that tether NOMPC to microtubules in mechanosensory neurons. Moreover, microtubule association is required for NOMPC mechanogating. Importantly, transferring the NOMPC ARs to mechanoinsensitive voltage-gated potassium channels confers mechanosensitivity to the chimeric channels. These experiments strongly support a tether mechanism of mechanogating for the NOMPC channel, providing insights into the basis of mechanosensitivity of mechanotransduction channels. 

Dewar, A. D., Wystrach, A., Graham, P. and Philippides, A. (2015). Navigation-specific neural coding in the visual system of Drosophila. Biosystems [Epub ahead of print]. PubMed ID: 26310914
Receptive fields have beed described for two classes of visually responsive neurons (R2 and R3/R4d ring neurons in the central complex) that are essential for visual tasks such as orientation memory for salient objects and simple pattern discriminations. What is interesting is that these cells have very large receptive fields and are very small in number, suggesting that each sub-population of cells might be a bottleneck in the processing of visual information for a specific behaviour, as each subset of cells effectively condenses information from approximately 3000 visual receptors in the eye, to fewer than 50 neurons in total. It has recently been shown how R1 ring neurons, which receive input from the same areas as the R2 and R3/R4d cells, are necessary for place learning in Drosophila. However, how R1 neurons enable place learning is unknown. By examining the information provided by different populations of hypothetical visual neurons in simulations of experimental arenas, this study shows that neurons with ring neuron-like receptive fields are sufficient for defining a location visually. In this way a link is provided between the type of information conveyed by ring neurons and the behaviour they support.

Yasunaga, K., Tezuka, A., Ishikawa, N., Dairyo, Y., Togashi, K., Koizumi, H. and Emoto, K. (2015). Adult Drosophila sensory neurons specify dendritic territories independently of dendritic contacts through the Wnt5-Drl signaling pathway. Genes Dev 29: 1763-1775. PubMed ID: 26302791
Sensory neurons with common functions are often nonrandomly arranged and form dendritic territories in stereotypic spatial patterns throughout the nervous system, yet molecular mechanisms of how neurons specify dendritic territories remain largely unknown. In Drosophila larvae, dendrites of class IV sensory (C4da) neurons completely but nonredundantly cover the whole epidermis, and the boundaries of these tiled dendritic fields are specified through repulsive interactions between homotypic dendrites. This study reports that, unlike the larval C4da neurons, adult C4da neurons rely on both dendritic repulsive interactions and external positional cues to delimit the boundaries of their dendritic fields. Wnt5 derived from sternites, the ventral-most part of the adult abdominal epidermis, were defined as the critical determinant for the ventral boundaries. Further genetic data indicate that Wnt5 promotes dendrite termination on the periphery of sternites through the Ryk receptor family kinase Derailed (Drl) and the Rho GTPase guanine nucleotide exchange factor Trio in C4da neurons. The findings thus uncover the dendritic contact-independent mechanism that is required for dendritic boundary specification and suggest that combinatory actions of the dendritic contact-dependent and -independent mechanisms may ensure appropriate dendritic territories of a given neuron.

Pulver, S. R., Bayley, T. G., Taylor, A. L., Berni, J., Bate, M. and Hedwig, B. (2015). Imaging fictive locomotor patterns in larval Drosophila. J Neurophysiol: jn 00731 02015. PubMed ID: 26311188
A preparation has been established in larval Drosophila to monitor fictive locomotion simultaneously across abdominal and thoracic segments of the isolated CNS using genetically encoded Ca2+ indicators. The Ca2+ signals closely followed spiking activity measured electrophysiologically in nerve roots. Three motor patterns are analyzed. Two comprise waves of Ca2+ signals which progress along the longitudinal body axis in a posterior-to-anterior or anterior-to-posterior direction. These waves had statistically indistinguishable inter-segmental phase delays compared to segmental contractions during forward and backward crawling behavior, despite being around 10 times slower. During these waves, motor neurons of the dorsal longitudinal and transverse muscles were active in the same order as the muscle groups are recruited during crawling behavior. A third fictive motor pattern exhibits a left-right asymmetry across segments and bears similarities with turning behavior in intact larvae, occurring equally frequently and involving asymmetry in the same segments. Ablation of the segments in which forward and backward waves of Ca2+ signals were normally initiated did not eliminate production of Ca2+ waves. When the brain and SOG were removed, the remaining ganglia retained the ability to produce both forward and backward waves of motor activity, although the speed and frequency of waves changed. Bilateral asymmetry of activity was reduced when the brain was removed, and abolished when the SOG was removed. This work paves the way to study the neural and genetic underpinnings of segmentally coordinated motor pattern generation in Drosophila using imaging techniques.

Sunday, September 20th

Garlena, R.A., Lennox, A.L., Baker, L.R., Parsons, T.E., Weinberg, S.M. and Stronach, B.E. (2015). Pvr receptor tyrosine kinase promotes tissue closure by coordinating corpse removal and epidermal zippering. Development [Epub ahead of print]. PubMed ID: 26293306
A leading cause of human birth defects is the incomplete fusion of tissues, often manifested in the palate, heart, or neural tube. To investigate the molecular control of tissue fusion, embryonic dorsal closure and pupal thorax closure in Drosophila are useful experimental models. This study finds that Pvr mutants have defects in dorsal midline closure with incomplete amnioserosa internalization and epidermal zippering, as well as cardia bifida. These defects are relatively mild in comparison to those seen with other signaling mutants such as the JNK pathway, and it was demonstrated that JNK signaling is not perturbed by altering Pvr receptor tyrosine kinase activity. Rather, modulation of Pvr levels in the ectoderm has an impact on PIP3 membrane accumulation consistent with a link to PI3K signal transduction. Polarized PI3K activity influences protrusive activity from the epidermal leading edge and protrusion area changes in accord with Pvr signaling intensity, providing a possible mechanism to explain Pvr mutant phenotypes. Tissue specific rescue experiments indicate a partial requirement in epithelial tissue, but confirm the essential role of Pvr in the hemocytes for embryonic survival. Taken together, the study argues that inefficient removal of the internalizing amnioserosa tissue by mutant hemocytes coupled with impaired midline zippering of mutant epithelium creates a situation in some embryos where dorsal midline closure is incomplete. Based on these observations, the study suggests that efferocytosis (corpse clearance) could contribute to proper tissue closure and thus may underlie some congenital birth defects.

Deng, S., Bothe, I. and Baylies, M. K. (2015). The formin Diaphanous regulates myoblast fusion through actin polymerization and Arp2/3 regulation. PLoS Genet 11: e1005381. PubMed ID: 26295716
The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3 (see Arpc1), its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. This study finds that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, this study demonstrates that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.

Wang, C., Guo, X., Dou, K., Chen, H. and Xi, R. (2015). Ttk69 acts as a master repressor of enteroendocrine cell specification in Drosophila intestinal stem cell lineages. Development [Epub ahead of print]. PubMed ID: 26293304
In adult Drosophila midgut, intestinal stem cells (ISCs) periodically produce progenitor cells that undergo a binary fate choice determined primarily by the levels of Notch activity they receive, before terminally differentiating into enterocytes (ECs) or enteroendocrine cells (EEs). This study identified Ttk69, a BTB domain-containing transcriptional repressor, as a master repressor of EE cell specification in the ISC lineages. Depletion of ttk69 in progenitor cells induces ISC proliferation and renders all committed progenitor cells to adopt EE cell specification, leading to the production of supernumerary EE cells in the intestinal epithelium. Conversely, forced expression of Ttk69 in progenitor cells is sufficient to prevent EE cell specification. The expression of Ttk69 is not regulated by Notch signaling, and forced activation of Notch, which is sufficient to induce EC specification of normal progenitor cells, fails to prevent EE cell specification of Ttk69-depleted progenitor cells. It was found that loss of Ttk69 leads to derepression of acheate-scute complex (AS-C) genes scute and asense, which then induce prospero expression to promote EE cell specification. These studies suggest that Ttk69 functions in parallel with Notch signaling and acts as a master repressor of EE cell specification in Drosophila ISC lineages primarily by suppressing AS-C genes.

Cheung, D. and Ma, J. (2015). Probing the impact of temperature on molecular events in a developmental system. Sci Rep 5: 13124. PubMed ID: 26286011
A well-appreciated general feature of development is the ability to achieve a normal outcome despite the inevitable variability at molecular, genetic, or environmental levels. But it is not well understood how changes in a global factor such as temperature bring about specific challenges to a developmental system in molecular terms. This question was addressed using early Drosophila embryos where the maternal gradient Bicoid (Bcd) instructs anterior-patterning (AP) patterning. Temperature can impact the amplitude of the Bcd gradient in the embryo. To evaluate how molecular decisions are made at different temperatures, Bcd concentrations and the expression of its target gene hunchback (hb) were quantified in individual embryos. The results suggest a relatively robust Bcd concentration threshold in inducing hb transcription within a temperature range. The results also reveal a complex nature of the effects of temperature on the progressions of developmental and molecular events of the embryo. This study thus advances the concept of developmental robustness by quantitatively elaborating specific features and challenges-imposed by changes in temperature-that an embryo must resolve.

Saturday, September 19th

Romano, G., Appocher, C., Scorzeto, M., Klima, R., Baralle, F. E., Megighian, A. and Feiguin, F. (2015). Glial TDP-43 regulates axon wrapping, GluRIIA clustering and fly motility by autonomous and non-autonomous mechanisms. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26276811
Alterations in the glial function of TDP-43 are becoming increasingly associated with the neurological symptoms observed in Amyotrophic Lateral Sclerosis (ALS), however, the physiological role of this protein in the glia or the mechanisms that may lead to neurodegeneration are unknown. To address these issues, the expression levels of TDP-43 was modulated in Drosophila glia; the protein was found to be required to regulate the subcellular wrapping of motoneuron axons, promote synaptic growth and the formation of glutamate receptor clusters at the neuromuscular junctions. Interestingly, it was determined that the glutamate transporter EAAT1 mediates the regulatory functions of TDP-43 in the glia, and genetic or pharmacological compensations of EAAT1 activity were demonstrated to be sufficient to modulate glutamate receptor clustering and locomotive behaviors in flies. The data uncovers autonomous and non-autonomous functions of TDP-43 in glia and suggests new experimentally based therapeutic strategies in ALS.

Sujkowski, A., Bazzell, B., Carpenter, K., Arking, R. and Wessells, R. J. (2015). Endurance exercise and selective breeding for longevity extend Drosophila healthspan by overlapping mechanisms. Aging (Albany NY) 7: 535-552. PubMed ID: 26298685
This study compared gene expression changes during endurance training in Drosophila melanogaster to gene expression changes during selective breeding for longevity. Microarrays indicate that 65% of gene expression changes found in flies selectively bred for longevity are also found in flies subjected to three weeks of exercise training. Both selective breeding and endurance training increase endurance, cardiac performance, running speed, flying height, and levels of autophagy in adipose tissue. Both interventions generally upregulate stress defense, folate metabolism, and lipase activity, while downregulating carbohydrate metabolism and odorant receptor expression. Several members of the methuselah-like (mthl) gene family are downregulated by both interventions. Knockdown of mthl-3 was sufficient to provide extension of negative geotaxis behavior, endurance and cardiac stress resistance. These results provide support for endurance exercise as a broadly acting anti-aging intervention and confirm that exercise training acts in part by targeting longevity assurance pathways.

Petruccelli, E., Lansdon, P. and Kitamoto, T. (2015). Exaggerated nighttime sleep and defective sleep homeostasis in a Drosophila knock-in model of human epilepsy. PLoS One 10: e0137758. PubMed ID: 26361221
Despite an established link between epilepsy and sleep behavior, it remains unclear how specific epileptogenic mutations affect sleep and subsequently influence seizure susceptibility. Recently, a fly knock-in model of human generalized epilepsy has been established that exhibits febrile seizures plus (GEFS+), a wide-spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a disease-causing mutation in their voltage-gated sodium channel (VGSC) gene and display semidominant heat-induced seizing, likely due to reduced GABAergic inhibitory activity at high temperature. This study shows that at room temperature the GEFS+ mutation dominantly modifies sleep, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. GEFS+ mutant sleep phenotypes were more resistant to pharmacologic reduction of GABA transmission by carbamazepine (CBZ) than controls, and were mitigated by reducing GABAA receptor expression specifically in wake-promoting pigment dispersing factor (PDF) neurons. These findings are consistent with increased GABAergic transmission to PDF neurons being mainly responsible for the enhanced nighttime sleep of GEFS+ mutants. This study reveals the sleep architecture of a Drosophila VGSC mutant that harbors a human GEFS+ mutation, and provides unique insight into the relationship between sleep and epilepsy.

O'Keefe, L. V., Lee, C. S., Choo, A. and Richards, R. I. (2015). Tumor Suppressor WWOX contributes to the elimination of tumorigenic cells in Drosophila melanogaster. PLoS One 10: e0136356. PubMed ID: 26302329
WWOX is a >1Mb gene spanning FRA16D Common Chromosomal Fragile Site, a region of DNA instability in cancer. Consequently, altered WWOX levels have been observed in a wide variety of cancers. In vitro studies have identified a large number and variety of potential roles for WWOX. Although its normal role in vivo and functional contribution to cancer have not been fully defined, WWOX does have an integral role in metabolism and can suppress tumor growth. Using Drosophila melanogaster as an in vivo model system, this study found that WWOX is a modulator of TNFalpha/Egr-mediated cell death. Altered levels of WWOX can modify phenotypes generated by low level ectopic expression of TNFalpha/Egr and this corresponds to altered levels of Caspase 3 activity. These results demonstrate an in vivo role for WWOX in promoting cell death. This form of cell death is accompanied by an increase in levels of reactive oxygen species, the regulation of can also be modified by altered WWOX activity. It is now hypothesized that, through regulation of reactive oxygen species, WWOX constitutes a link between alterations in cellular metabolism observed in cancer cells and their ability to evade normal cell death pathways. WWOX activity is required for the efficient removal of tumorigenic cells from a developing epithelial tissue. Together these results provide a molecular basis for the tumor suppressor functions of WWOX and the better prognosis observed in cancer patients with higher levels of WWOX activity. Understanding the conserved cellular pathways to which WWOX contributes provides novel possibilities for the development of therapeutic approaches to restore WWOX function in cancer.

Friday, September 18th

Oh, H., Slattery, M., Ma, L., White, K. P., Mann, R. S. and Irvine, K. D. (2014). Yorkie promotes transcription by recruiting a histone methyltransferase complex. Cell Rep 8: 449-459. PubMed ID: 25017066
Hippo signaling limits organ growth by inhibiting the transcriptional coactivator Yorkie. Despite the key role of Yorkie in both normal and oncogenic growth, the mechanism by which it activates transcription has not been defined. This paper reports that Yorkie binding to chromatin correlates with histone H3K4 methylation and is sufficient to locally increase it. Yorkie can recruit a histone methyltransferase complex through binding between WW domains of Yorkie and PPxY sequence motifs of NcoA6, a subunit of the Trithorax-related (Trr) methyltransferase complex. Cell culture and in vivo assays establish that this recruitment of NcoA6 contributes to Yorkie's ability to activate transcription. Mammalian NcoA6, a subunit of Trr-homologous methyltransferase complexes, can similarly interact with Yorkie's mammalian homolog YAP. These results implicate direct recruitment of a histone methyltransferase complex as central to transcriptional activation by Yorkie, linking the control of cell proliferation by Hippo signaling to chromatin modification.

Ghasemi, M., Pawar, H., Mishra, R. K. and Brahmachari, V. (2015). The functional diversity of Drosophila Ino80 in development. Mech Dev [Epub ahead of print]. PubMed ID: 26253267
Ino80 is well known as a chromatin remodeling protein with the catalytic function of DNA dependent ATPase and is highly conserved across phyla. Ino80 in human and Drosophila is known to form the Ino80 complex in association with the DNA binding protein Ying-Yang 1 (YY1)/Pleiohomeotic (Pho) the Drosophila homologue. Ino80 sub-family of proteins has two functional domains, namely, the DNA dependent ATPase and the DNA binding domain. In the background of the essential role of dIno80 in development, this study provides evidence of Pho independent function of dIno80 in development and analyzes the dual role of dIno80 in activation as well as repression in the context of the homeotic gene Scr (Sex combs reduced) in imaginal discs. This differential effect of dIno80 in different imaginal discs suggests the contextual function of dIno80 as an Enhancer of Trithorax and Polycomb (ETP). The study speculates on the role of dIno80 as a chromatin remodeler on one hand and a potential recruiter of epigenetic regulatory complexes on the other.

Wolle, D., Cleard, F., Aoki, T., Deshpande, G., Schedl, P. and Karch, F. (2015). Functional requirements for Fab-7 boundary activity in the Bithorax Complex. Mol Cell Biol. PubMed ID: 26303531
Chromatin boundaries are architectural elements that determine the 3-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity The Fab-7 boundary from the Drosophila Bithorax complex (BX-C) is required for the parasegment specific expression of the Abd-B gene. This study used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and a novel approximately 700kD complex, the LBC, is described that binds to Fab-7 sequences that have insulator function in late embryos and adults. The LBC is enriched in nuclear extracts from late but not early embryos, and it contains three insulator proteins, GAF, Mod(mdg4) and E(y)2. Its DNA bindings properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display little sequence similarities. Finally it was shown that mutations that abrogate LBC binding in vitro inactivate the Fab-7 boundary in BX-C.

Hill, H. and Golic, K. G. (2015). Preferential breakpoints in the recovery of broken dicentric chromosomes in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 26294667
A system was designed to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly-euploid chromosome. It was expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half of the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the 'Laird hypothesis' that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including Fragile X. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.

Thursday, September 17th

Oh, S., Kato, M., Zhang, C., Guo, Y. and Beachy, P. A. (2015). A comparison of Ci/Gli activity as regulated by Sufu in Drosophila and mammalian hedgehog response. PLoS One 10: e0135804. PubMed ID: 26271100
Suppressor of fused [Su(fu)/Sufu], one of the most conserved components of the Hedgehog (Hh) signaling pathway, binds Ci/Gli transcription factors and impedes activation of target gene expression. In Drosophila, the Su(fu) mutation has a minimal phenotype, and this study shows that Ci transcriptional activity in large part is regulated independently of Su(fu) by other pathway components. Mutant mice lacking Sufu in contrast show excessive pathway activity and die as embryos with patterning defects. In cultured cells Hh stimulation can augment transcriptional activity of a Gli2 variant lacking Sufu interaction and, surprisingly, regulation of Hh pathway targets is nearly normal in the neural tube of Sufu-/- mutant embryos that also lack Gli1 function. Some degree of Hh-induced transcriptional activation of Ci/Gli thus can occur independently of Sufu in both flies and mammals. It is further noted that Sufu loss can also reduce Hh induction of high-threshold neural tube fates, such as floor plate, suggesting a possible positive pathway role for Sufu.

Zhou, Z., Xu, C., Chen, P., Liu, C., Pang, S., Yao, X. and Zhang, Q. (2015). Stability of HIB-Cul3 E3 ligase adaptor HIB is regulated by self-degradation and availability of its substrates. Sci Rep 5: 12709. PubMed ID: 26263855
The HIB-Cul3 complex E3 ligase regulates physiological homeostasis through regulating its substrate stability and its activity can be modulated by changing HIB abundance. However, regulation of HIB remains elusive. This study provides evidence that HIB is degraded through the proteasome by Cul3-mediated polyubiquitination in K48 manner in Drosophila. Strikingly, HIB is targeted for degradation by itself. This study further identify that three degrons and K185 and K198 of HIB are essential for its auto-degradation. Finally, it was demonstrated that HIB-Cul3 substrates, Ci and Puc, can effectively protect HIB from HIB-Cul3-mediated degradation. Taken together, this study indicates that there is an exquisite equilibrium between the adaptor and targets to achieve the tight control of the HIB, which is essential for maintaining suitable Hh and JNK signaling. And the mechanism of adaptor self-degradation and reciprocal control of the abundance between adaptor and its substrates is also applied to BTB-Cul3 E3 ligase adaptor dKeap1, dDiablo and dKLHL18.

Marada, S., Navarro, G., Truong, A., Stewart, D. P., Arensdorf, A. M., Nachtergaele, S., Angelats, E., Opferman, J. T., Rohatgi, R., McCormick, P. J. and Ogden, S. K. (2015). Functional divergence in the role of N-linked glycosylation in Smoothened signaling. PLoS Genet 11: e1005473. PubMed ID: 26291458
The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Galphai. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. This study presents a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Galphai, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, a profound shift is postulated in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.

Monahan, A. J. and Starz-Gaiano, M. (2015). Socs36E limits STAT signaling via Cullin2 and a SOCS-box independent mechanism in the Drosophila egg chamber. Mech Dev [Epub ahead of print]. PubMed ID: 26277564
The Suppressor of Cytokine Signaling (SOCS) proteins are critical, highly conserved feedback inhibitors of signal transduction cascades. The family of SOCS proteins is divided into two groups: ancestral and vertebrate-specific SOCS proteins. Vertebrate-specific SOCS proteins have been heavily studied as a result of their strong mutant phenotypes. However, the ancestral clade remains less studied, a potential result of genetic redundancies in mammals. Use of the genetically tractable organism Drosophila melanogaster enables in vivo assessment of signaling components and mechanisms with less concern about the functional redundancy observed in mammals. This study investigated how the SOCS family member Suppressor of Cytokine Signaling at 36E (Socs36E) attenuates Jak/STAT activation during specification of motile border cells in Drosophila oogenesis. Socs36E genetically interacts with the Cullin2 (Cul2) scaffolding protein. Like Socs36E, Cul2 is required to limit the number of motile cells in egg chambers. Loss of Cul2 in the follicle cells significantly increased nuclear STAT protein levels, which resulted in additional cells acquiring invasive properties. Further, reduction of Cul2 suppressed border cell migration defects that occur in a Stat92E-sensitized genetic background. These data incorporated Cul2 into a previously described Jak/STAT-directed genetic regulatory network that is required to generate a discrete boundary between cell fates. It was also found that Socs36E is able to attenuate STAT activity in the egg chamber when it does not have a functional SOCS box. Collectively, this work contributes mechanistic insight to a Jak/STAT regulatory genetic circuit, and suggests that Socs36E regulates Jak/STAT signaling via a Cul2-dependent mechanism, as well as by a Cullin-independent manner, in vivo.

Wednesday, September 16th

Fiedler, M., Graeb, M., Mieszczanek, J., Rutherford, T. J., Johnson, C. M. and Bienz, M. (2015). An ancient Pygo-dependent Wnt enhanceosome integrated by Chip/LDB-SSDP. Elife 4. PubMed ID: 26312500
TCF/LEF factors (see Drosophila Pangolin) are ancient context-dependent enhancer-binding proteins that are activated by β-catenin (see Drosophila Armadillo) following Wnt signaling. They control embryonic development and adult stem cell compartments, and their dysregulation often causes cancer. β-catenin-dependent transcription relies on the NPF motif of Pygo proteins. This study used a proteomics approach to discover the Chip/LDB-SSDP (ChiLS) complex as the ligand specifically binding to NPF. ChiLS also recognizes NPF motifs in other nuclear factors including Runt/RUNX2 and Drosophila ARID1, and binds to Groucho/TLE. Studies of Wnt-responsive dTCF enhancers in the Drosophila embryonic midgut indicate how these factors interact to form the Wnt enhanceosome, primed for Wnt responses by Pygo. Together with previous evidence, this study indicates that ChiLS confers context-dependence on TCF/LEF by integrating multiple inputs from lineage and signal-responsive factors, including enhanceosome switch-off by Notch. Its pivotal function in embryos and stem cells explain why its integrity is crucial in the avoidance of cancer.

Wunderlich, Z., Bragdon, M.D., Vincent, B.J., White, J.A., Estrada, J. and DePace, A.H. (2015). Krüppel expression levels are maintained through compensatory evolution of shadow enhancers. Cell Rep [Epub ahead of print]. PubMed ID: 26344774
Many developmental genes are controlled by shadow enhancers-pairs of enhancers that drive overlapping expression patterns. This study hypothesized that compensatory evolution can maintain the total expression of a gene, while individual shadow enhancers diverge between species. This study analyzed expression driven by orthologous pairs of shadow enhancers from Drosophila melanogaster, Drosophila yakuba, and Drosophila pseudoobscura that control expression of Krüppel, a transcription factor that patterns the anterior-posterior axis of blastoderm embryos. The expression driven by the pair of enhancers was conserved between these three species, but expression levels driven by the individual enhancers was not. Using sequence analysis and experimental perturbation, it was shown that each shadow enhancer is regulated by different transcription factors. These results support the hypothesis that compensatory evolution can occur between shadow enhancers, which has implications for mechanistic and evolutionary studies of gene regulation

Nachman, A., Halachmi, N., Matia, N., Manzur, D. and Salzberg, A. (2015).. Deconstructing the complexity of regulating common properties in different cell types: lessons from the delilah gene. Dev Biol 403: 180-191. PubMed ID: 25989022
To decode how adhesion is regulated in cells stemming from different pedigrees this study analyzed the regulatory region that drives the expression of Delilah, which is a transcription factor that serves as a central determinant of cell adhesion, particularly by inducing expression of βPS-integrin. Activation of dei transcription was shown to be mediated through multiple cis regulatory modules, each driving transcription in a spatially and temporally restricted fashion. Thus the dei gene provides a molecular platform through which cell adhesion can be regulated at the transcriptional level in different cellular milieus. Moreover, these regulatory modules respond, often directly, to central regulators of cell identity in each of the dei-expressing cell types, such as D-Mef2 in muscle cells, Stripe in tendon cells and Blistered in wing intervein cells. These findings suggest that the acquirement of common cellular properties shared by different cell types is embedded within the unique differentiation program dictated to each of these cells by the major determinants of its identity.

Kok, K., Ay, A., Li, L. M. and Arnosti, D. N. (2015). Genome-wide errant targeting by Hairy. Elife 4 [Epub ahead of print]. PubMed ID: 26305409
Metazoan transcriptional repressors regulate chromatin through diverse histone modifications. Contributions of individual factors to the chromatin landscape in development is difficult to establish, as global surveys reflect multiple changes in regulators. Therefore, the conserved Hairy/Enhancer of Split family repressor Hairy was studied, analyzing histone marks and gene expression in Drosophila embryos. This long-range repressor mediates histone acetylation and methylation in large blocks, with highly context-specific effects on target genes. Most strikingly, Hairy exhibits biochemical activity on many loci that are uncoupled to changes in gene expression. Rather than representing inert binding sites, as suggested for many eukaryotic factors, many regions are targeted errantly by Hairy to modify the chromatin landscape. these findings emphasize that identification of active cis-regulatory elements must extend beyond the survey of prototypical chromatin marks. It is speculated that this errant activity may provide a path for creation of new regulatory elements, facilitating the evolution of novel transcriptional circuits.

Tuesday, September 15th

Williams, J., Boin, N. G., Valera, J. M. and Johnson, A. N. (2015). Noncanonical roles for Tropomyosin during myogenesis. Development [Epub ahead of print]. PubMed ID: 26293307
For skeletal muscle to produce movement, individual myofibers must form stable contacts with tendon cells and then assemble sarcomeres. The myofiber precursor is the nascent myotube, and during myogenesis the myotube completes guided elongation to reach its target tendons. Unlike the well-studied events of myogenesis, such as myoblast specification and myoblast fusion, the molecules that regulate myotube elongation are largely unknown. In Drosophila, hoi polloi (hoip) encodes a highly-conserved RNA binding protein and hoip mutant embryos are largely paralytic due to defects in myotube elongation and sarcomeric protein expression. The hoip mutant background was used as a platform to identify novel regulators of myogenesis, and surprising developmental functions were uncovered for the sarcomeric protein Tropomyosin 2 (Tm2). Hoip responsive sequences were identified in the coding region of the Tm2 mRNA that are essential for Tm2 protein expression in developing myotubes. Tm2 overexpression rescued the hoip myogenic phenotype by promoting F-actin assembly at the myotube leading edge, by restoring the expression of additional sarcomeric RNAs, and by promoting myoblast fusion. Embryos that lack Tm2 also showed reduced sarcomeric protein expression, and embryos that expressed a gain-of-function Tm2 allele showed both fusion and elongation defects. Tropomyosin therefore dictates fundamental steps of myogenesis prior to regulating contraction in the sarcomere.

Stoiber, M. H., Olson, S., May, G. E., Duff, M. O., Manent, J., Obar, R., Guruharsha, K., Artavanis-Tsakonas, S., Brown, J. B., Graveley, B. R. and Celniker, S. E. (2015). Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila. Genome Res [Epub ahead of print]. PubMed ID: 26294687
In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. Twenty distinct RNA binding proteins (RBPs) were immunopurified from cultured Drosophila melanogaster cells under native conditions, and both the RNA and protein compositions of these RNP complexes were determined. "High occupancy target" (HOT) RNAs were identified that interact with the majority of the RBPs surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery as well as RNA binding and translation initiation proteins. The RNP complexes contain proteins and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultra-complex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). This data is consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. From the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs this study identified extensive cross-regulatory and hierarchical interactions in post-transcriptional control.

Brooks, A. N., Duff, M. O., May, G., Yang, L., Bolisetty, M., Landolin, J., Wan, K., Sandler, J., Celniker, S. E., Graveley, B. R. and Brenner, S. E. (2015). Regulation of alternative splicing in Drosophila by 56 RNA binding proteins. Genome Res [Epub ahead of print]. PubMed ID: 26294686
Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. This study used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. A cross-regulatory network was identified where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

Senti, K. A., Jurczak, D., Sachidanandam, R. and Brennecke, J. (2015). piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire. Genes Dev 29: 1747-1762. PubMed ID: 26302790
PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound approximately 23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while secondary piRNA biogenesis depends on piRNA-guided transcript cleavage and results in piRNA amplification. This study analyzed the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. Secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts-including those from piRNA clusters-as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, these findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways.

Xiong, X. P., Vogler, G., Kurthkoti, K., Samsonova, A. and Zhou, R. (2015). SmD1 modulates the miRNA pathway independently of its pre-mRNA splicing function. PLoS Genet 11: e1005475. PubMed ID: 26308709
microRNAs (miRNAs) are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs) that contain Argonaute (AGO) family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. This study shows that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP) implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. This study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be evolutionarily widespread.

Peter, D., Weber, R., Kone, C., Chung, M. Y., Ebertsch, L., Truffault, V., Weichenrieder, O., Igreja, C. and Izaurralde, E. (2015). Mextli proteins use both canonical bipartite and novel tripartite binding modes to form eIF4E complexes that display differential sensitivity to 4E-BP regulation. Genes Dev 29: 1835-1849. PubMed ID: 26294658
The eIF4E-binding proteins (4E-BPs) are a diverse class of translation regulators that share a canonical eIF4E-binding motif (4E-BM) with eIF4G. Consequently, they compete with eIF4G for binding to eIF4E, thereby inhibiting translation initiation. Mextli (Mxt) is an unusual 4E-BP that promotes translation by also interacting with eIF3. This study presents the crystal structures of the eIF4E-binding regions of the Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) Mxt proteins in complex with eIF4E in the cap-bound and cap-free states. The structures reveal unexpected evolutionary plasticity in the eIF4E-binding mode, with a classical bipartite interface for Ce Mxt and a novel tripartite interface for Dm Mxt. Both interfaces comprise a canonical helix and a noncanonical helix that engage the dorsal and lateral surfaces of eIF4E, respectively. Remarkably, Dm Mxt contains a C-terminal auxiliary helix that lies anti-parallel to the canonical helix on the eIF4E dorsal surface. In contrast to the eIF4G and Ce Mxt complexes, the Dm eIF4E-Mxt complexes are resistant to competition by bipartite 4E-BPs, suggesting that Dm Mxt can bind eIF4E when eIF4G binding is inhibited. These results uncovered unexpected diversity in the binding modes of 4E-BPs, resulting in eIF4E complexes that display differential sensitivity to 4E-BP regulation.

Monday, September 14th

Barrios, N., González-Pérez, E., Hernández, R. and Campuzano, S. (2015). The homeodomain Iroquois proteins control cell cycle progression and regulate the size of developmental fields. PLoS Genet 11: e1005463. PubMed ID: 26305360
During development, proper differentiation and final organ size rely on the control of territorial specification and cell proliferation. Although many regulators of these processes have been identified, how both are coordinated remains largely unknown. The homeodomain Iroquois/Irx proteins play a key, evolutionarily conserved, role in territorial specification. This study shows that in the imaginal discs, reduced function of Iroquois genes promotes cell proliferation by accelerating the G1 to S transition. Conversely, their increased expression causes cell-cycle arrest, down-regulating the activity of the Cyclin E/Cdk2 complex. It was demonstrated that physical interaction of the Iroquois protein Caupolican with Cyclin E-containing protein complexes, through its IRO box and Cyclin-binding domains, underlies its activity in cell-cycle control. Thus, Drosophila Iroquois proteins are able to regulate cell-autonomously the growth of the territories they specify. Moreover, the study provides a molecular mechanism for a role of Iroquois/Irx genes as tumour suppressors

Organista, M. F., Martin, M., de Celis, J. M., Barrio, R., Lopez-Varea, A., Esteban, N., Casado, M. and de Celis, J. F. (2015). The Spalt transcription factors generate the transcriptional landscape of the Drosophila melanogaster wing pouch central region. PLoS Genet 11: e1005370. PubMed ID: 26241320
The Drosophila genes spalt major (salm) and spalt-related (salr) encode Zn-finger transcription factors regulated by the Decapentaplegic (Dpp) signalling pathway in the wing imaginal disc. To identify candidate Salm/Salr target genes, the expression profile of salm/salr knockdown wing discs was compared with control discs in microarray experiments. In situ hybridization was used to study the expression pattern of the genes whose mRNA levels varied significantly, and a complex transcription landscape was uncovered regulated by the Spalt proteins in the wing disc. Interestingly, candidate Salm/Salr targets include genes which expression is turned off and genes which expression is positively regulated by Salm/Salr. Furthermore, loss-of-function phenotypic analysis of these genes indicates, for a fraction of them, a requirement for wing growth and patterning. The identification and analysis of candidate Salm/Salr target genes opens a new avenue to reconstruct the genetic structure of the wing, linking the activity of the Dpp pathway to the development of this epithelial tissue.

Wang, Y., Antunes, M., Anderson, A. E., Kadrmas, J. L., Jacinto, A. and Galko, M. J. (2015). Integrin adhesions suppress syncytium formation in the Drosophila larval epidermis. Curr Biol 25: 2215-2227. PubMed ID: 26255846
Integrins are critical for barrier epithelial architecture. Integrin loss in vertebrate skin leads to blistering and wound healing defects. However, how integrins and associated proteins maintain the regular morphology of epithelia is not well understood. This study found that targeted knockdown of the integrin focal adhesion (FA) complex components β-integrin, PINCH, and integrin-linked kinase (ILK) caused formation of multinucleate epidermal cells within the Drosophila larval epidermis. This phenotype was specific to the integrin FA complex and not due to secondary effects on polarity or junctional structures. The multinucleate cells resembled the syncytia caused by physical wounding. Live imaging of wound-induced syncytium formation in the pupal epidermis suggested direct membrane breakdown leading to cell-cell fusion and consequent mixing of cytoplasmic contents. Activation of Jun N-terminal kinase (JNK) signaling, which occurs upon wounding, also correlated with syncytium formation induced by PINCH knockdown. Further, ectopic JNK activation directly caused epidermal syncytium formation. No mode of syncytium formation, including that induced by wounding, genetic loss of FA proteins, or local JNK hyperactivation, involved misregulation of mitosis or apoptosis. Finally, the mechanism of epidermal syncytium formation following JNK hyperactivation and wounding appeared to be direct disassembly of FA complexes. In conclusion, the loss-of-function phenotype of integrin FA components in the larval epidermis resembles a wound. Integrin FA loss in mouse and human skin also causes a wound-like appearance. The results reveal a novel and unexpected role for proper integrin-based adhesion in suppressing larval epidermal cell-cell fusion-a role that may be conserved in other epithelia.

Ferreira, A. and Milan, M. (2015). Dally proteoglycan mediates the autonomous and nonautonomous effects on tissue growth caused by activation of the PI3K and TOR pathways. PLoS Biol 13: e1002239. PubMed ID: 26313758
How cells acquiring mutations in tumor suppressor genes outcompete neighboring wild-type cells is poorly understood. The PTEN and TSC-TOR pathways are frequently activated in human cancer, and this activation is often causative of tumorigenesis. This study used the Gal4-UAS system in Drosophila imaginal primordia, highly proliferative and growing tissues, to analyze the impact of restricted activation of these pathways on neighboring wild-type cell populations. Activation of these pathways leads to an autonomous induction of tissue overgrowth and to a remarkable nonautonomous reduction in growth and proliferation rates of adjacent cell populations. This nonautonomous response occurs independently of where these pathways are activated, is functional all throughout development, takes place across compartments, and is distinct from cell competition. The observed autonomous and nonautonomous effects on tissue growth rely on the up-regulation of the proteoglycan Dally, a major element involved in modulating the spreading, stability, and activity of the growth promoting Decapentaplegic (Dpp)/transforming growth factor β(TGF-β) signaling molecule. These findings indicate that a reduction in the amount of available growth factors contributes to the outcompetition of wild-type cells by overgrowing cell populations. During normal development, the PI3K/PTEN and TSC/TOR pathways play a major role in sensing nutrient availability and modulating the final size of any developing organ. This study presents evidence that Dally also contributes to integrating nutrient sensing and organ scaling, the fitting of pattern to size.

Sunday, September 13th

Kroll, J.R., Wong, K.G., Siddiqui, F.M. and Tanouye, M.A. (2015). Disruption of endocytosis with the dynamin mutant shibirets1 suppresses seizures in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 26341658
One challenge is to control epilepsies that do not respond to currently available medications. Since seizures consist of coordinated and high frequency neural activity, the goal of this study was to disrupt neurotransmission with a synaptic transmission mutant and evaluate its ability to suppress seizures. It was found that the mutant shibire, encoding Dynamin, suppresses seizure-like activity in multiple seizure-sensitive Drosophila genotypes, one of which resembles human intractable epilepsy in several aspects. Due to the requirement of Dynamin in endocytosis, increased temperature in the shits1 mutant causes impairment of synaptic vesicle recycling, and is associated with the suppression of the seizure-like activity. Additionally, the giant fiber neuron was identified to be critical in the seizure circuit that is sufficient to suppress seizures. Overall, these results implicate mutant Dynamin as an effective seizure suppressor, suggesting that targeting or limiting the availability of synaptic vesicles could be an effective and general method of controlling epilepsy disorders.

Yu, S., Jang, Y., Paik, D., Lee, E. and Park, J.J. (2015). Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 26319556
NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. This study found that ubiquitous Nmdmc overexpression enhances Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body is sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincide with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan.

Fernandez-Funez, P., Zhang, Y., Sanchez-Garcia, J., de Mena, L., Khare, S., Golde, T. E., Levites, Y. and Rincon-Limas, D. E. (2015). Anti-Aβ single-chain variable fragment antibodies exert synergistic neuroprotective activities in Drosophila models of Alzheimer's disease. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26253732
Both active and passive immunotherapy protocols decrease insoluble amyloid-ss42 (Ass42; see Drosophila Appl) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Ass42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. This study shows that scFvs against the N- and C-terminus of Ass42 (scFv9 and scFV42.2, respectively) that decrease insoluble Ass42 in CRND mice are neuroprotective in Drosophila models of Ass42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, co-expression of both anti-Ass scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Ass42 epitopes can be more effective than targeting a single epitope. Overall, this study demonstrates the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Ass scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities.

Shaw, J. L., Zhang, S. and Chang, K. T. (2015). Bidirectional regulation of Amyloid precursor protein-induced memory defects by Nebula/DSCR1: A protein upregulated in Alzheimer's disease and Down syndrome. J Neurosci 35: 11374-11383. PubMed ID: 26269644
Aging individuals with Down syndrome (DS) have an increased risk of developing Alzheimer's disease (AD), a neurodegenerative disorder characterized by impaired memory. Memory problems in both DS and AD individuals usually develop slowly and progressively get worse with age, but the cause of this age-dependent memory impairment is not well understood. This study examines the functional interactions between Down syndrome critical region 1 (DSCR1) and amyloid-precursor protein (APP), proteins upregulated in both DS and AD, in regulating memory. Using Drosophila as a model, this study found that overexpression of nebula (fly homolog of DSCR1) initially protects against APP-induced memory defects by correcting calcineurin and cAMP signaling pathways but accelerates the rate of memory loss and exacerbates mitochondrial dysfunction in older animals. Transient upregulation of Nebula/DSCR1 or acute pharmacological inhibition of calcineurin in aged flies protected against APP-induced memory loss. These data suggest that calcineurin dyshomeostasis underlies age-dependent memory impairments and further imply that chronic Nebula/DSCR1 upregulation may contribute to age-dependent memory impairments in AD in DS.

Saturday, September 12th

Albin, S.D., Kaun, K.R., Knapp, J.M., Chung, P., Heberlein, U. and Simpson, J.H. (2015). A subset of serotonergic neurons evokes hunger in adult Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 26344091
Hunger is a complex motivational state that drives multiple behaviors. The sensation of hunger is caused by an imbalance between energy intake and expenditure. Although progress is being made, how hunger is represented in the brain and how it coordinates these behavioral responses is not fully understood in any system. This study uses Drosophila melanogaster to identify neurons encoding hunger. A small group of neurons was found that, when activated, induces a fed fly to eat as though it is starved, suggesting that these neurons are downstream of the metabolic regulation of hunger. Artificially activating these neurons also promotes appetitive memory performance in sated flies, indicating that these neurons are not simply feeding command neurons but likely play a more general role in encoding hunger. The neurons relevant for the feeding effect are serotonergic and project broadly within the brain, suggesting a possible mechanism for how various responses to hunger are coordinated. These findings extend the understanding of the neural circuitry that drives feeding and enable future exploration of how state influences neural activity within this circuit.

Hampel, S., Franconville, R., Simpson, J.H. and Seeds, A.M. (2015). A neural command circuit for grooming movement control. Elife [Epub ahead of print]. PubMed ID: 26344548
Animals perform many stereotyped movements, but how nervous systems are organized for controlling specific movements remains unclear. This study used anatomical, optogenetic, behavioral, and physiological techniques to identify a circuit in Drosophila melanogaster that can elicit stereotyped leg movements that groom the antennae. Mechanosensory chordotonal neurons detect displacements of the antennae and excite three different classes of functionally connected interneurons, which include two classes of brain interneurons and different parallel descending neurons. This multilayered circuit is organized such that neurons within each layer are sufficient to specifically elicit antennal grooming. However, differences were found in the durations of antennal grooming elicited by neurons in the different layers, suggesting that the circuit is organized to both command antennal grooming and control its duration. As similar features underlie stimulus-induced movements in other animals, there is a possibility of a common circuit organization for movement control that can be dissected in Drosophila

Lemon, W. C., Pulver, S. R., Hockendorf, B., McDole, K., Branson, K., Freeman, J. and Keller, P. J. (2015). Whole-central nervous system functional imaging in larval Drosophila. Nat Commun 6: 7924. PubMed ID: 26263051
Understanding how the brain works in tight concert with the rest of the central nervous system (CNS) hinges upon knowledge of coordinated activity patterns across the whole CNS. This paper presents a method for measuring activity in an entire, non-transparent CNS with high spatiotemporal resolution. The method combines light-sheet microscope capable of simultaneous multi-view imaging at volumetric speeds 25-fold faster than the state-of-the-art, a whole-CNS imaging assay for the isolated Drosophila larval CNS and a computational framework for analysing multi-view, whole-CNS calcium imaging data. Both brain and ventral nerve cord were imaged, covering the entire CNS at 2 or 5 Hz with two- or one-photon excitation, respectively. By mapping network activity during fictive behaviours and quantitatively comparing high-resolution whole-CNS activity maps across individuals, functional connections are predicted between CNS regions and neurons in the brain are revealed that identify type and temporal state of motor programs executed in the ventral nerve cord.

Lebreton, S., Trona, F., Borrero-Echeverry, F., Bilz, F., Grabe, V., Becher, P. G., Carlsson, M. A., Nassel, D. R., Hansson, B. S., Sachse, S. and Witzgall, P. (2015). Feeding regulates sex pheromone attraction and courtship in Drosophila females. Sci Rep 5: 13132. PubMed ID: 26255707
In Drosophila melanogaster, gender-specific behavioural responses to the male-produced sex pheromone cis-vaccenyl acetate (cVA) rely on sexually dimorphic, third-order neural circuits. This study shows that nutritional state in female flies modulates cVA perception in first-order olfactory neurons. Starvation increases, and feeding reduces attraction to food odour, in both sexes. Adding cVA to food odour, however, maintains attraction in fed females, while it has no effect in males. Upregulation of sensitivity and behavioural responsiveness to cVA in fed females is paralleled by a strong increase in receptivity to male courtship. Functional imaging of the antennal lobe (AL), the olfactory centre in the insect brain, shows that olfactory input to DA1 and VM2 glomeruli is also modulated by starvation. Knocking down insulin receptors in neurons converging onto the DA1 glomerulus suggests that insulin-signalling partly controls pheromone perception in the AL, and adjusts cVA attraction according to nutritional state and sexual receptivity in Drosophila females.

Friday, September 11th

Barckmann, B., Pierson, S., Dufourt, J., Papin, C., Armenise, C., Port, F., Grentzinger, T., Chambeyron, S., Baronian, G., Desvignes, J. P., Curk, T. and Simonelig, M. (2015). Aubergine iCLIP reveals piRNA-dependent decay of mRNAs involved in germ cell development in the early embryo. Cell Rep 12: 1205-1216. PubMed ID: 26257181
The Piwi-interacting RNA (piRNA) pathway plays an essential role in the repression of transposons in the germline. Other functions of piRNAs such as post-transcriptional regulation of mRNAs are now emerging. This study perform iCLIP with the PIWI protein Aubergine (Aub) and identified hundreds of maternal mRNAs interacting with Aub in the early Drosophila embryo. Gene expression profiling reveals that a proportion of these mRNAs undergo Aub-dependent destabilization during the maternal-to-zygotic transition. Strikingly, Aub-dependent unstable mRNAs encode germ cell determinants. iCLIP with an Aub mutant that is unable to bind piRNAs confirms piRNA-dependent binding of Aub to mRNAs. Base pairing between piRNAs and mRNAs can induce mRNA cleavage and decay that are essential for embryonic development. These results suggest general regulation of maternal mRNAs by Aub and piRNAs, which plays a key developmental role in the embryo through decay and localization of mRNAs encoding germ cell determinants.

Yang, N., Yu, Z., Hu, M., Wang, M., Lehmann, R. and Xu, R.M. (2015). Structure of Drosophila Oskar reveals a novel RNA binding protein. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26324911
Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. This study reports the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix-fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, it was found that Osk-C binds the 3'UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3'UTRs, and provide structural insights into a novel protein-RNA interaction motif involving a hydrolase-related domain.

Yan, D. and Perrimon, N. (2015). spenito is required for sex determination in Drosophila melanogaster. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26324914
Sex-lethal (Sxl) encodes the master regulator of the sex determination pathway in Drosophila and acts by controlling sex identity in both soma and germ line. In females Sxl maintains its own expression by controlling the alternative splicing of its own mRNA. This study identifies a novel sex determination gene, spenito (nito) that encodes a SPEN family protein. Loss of nito activity results in stem cell tumors in the female germ line as well as female-to-male somatic transformations. It was shown that Nito is a ubiquitous nuclear protein that controls the alternative splicing of the Sxl mRNA by interacting with Sxl protein and pre-mRNA, suggesting that it is directly involved in Sxl auto-regulation. Given that SPEN family proteins are frequently mutated in cancers, these results suggest that these factors might be implicated in tumorigenesis through splicing regulation.

Shen, S., et al. (2015). A miR-130a-YAP positive feedback loop promotes organ size and tumorigenesis. Cell Res 25: 997-1012. PubMed ID: 26272168
Organ size determination is one of the most intriguing unsolved mysteries in biology. Aberrant activation of the major effector and transcription co-activator YAP (see Drosophila Yorkie) in the Hippo pathway causes drastic organ enlargement in development. This study reports that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4 (a vestigial homolog), an inhibitor of YAP activity, thereby amplifying the YAP signals. Inhibition of miR-130a reversed liver size enlargement induced by Hippo pathway inactivation and blocked YAP-induced tumorigenesis. Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog Tondu-domain-containing Growth Inhibitor (SdBP/Tgi). These findings reveal an evolutionarily conserved positive feedback mechanism underlying robustness of the Hippo pathway in size control and tumorigenesis.

Schachtner, L. T., Sola, I. E., Forand, D., Antonacci, S., Postovit, A. J., Mortimer, N. T., Killian, D. J. and Olesnicky, E. C. (2015). Drosophila Shep and C. elegans SUP-26 are RNA-binding proteins that play diverse roles in nervous system development. Dev Genes Evol [Epub ahead of print]. PubMed ID: 26271810
The Caenorhabditis elegans gene sup-26 encodes a well-conserved RNA-recognition motif-containing RNA-binding protein (RBP) that functions in dendrite morphogenesis of the PVD sensory neuron. The Drosophila ortholog of sup-26, alan shepard (shep), is expressed throughout the nervous system and has been shown to regulate neuronal remodeling during metamorphosis. These studies were extended to show that sup-26 and shep are required for the development of diverse cell types within the nematode and fly nervous systems during embryonic and larval stages. Roles are described for sup-26 in regulating dendrite number and the expression of genes involved in mechanosensation within the nematode peripheral nervous system. In Drosophila, shep regulates dendrite length and branch order of nociceptive neurons, regulates the organization of neuronal clusters of the peripheral nervous system and the organization of axons within the ventral nerve cord. Taken together, these results suggest that shep/sup-26 orthologs play diverse roles in neural development across animal species. Moreover, potential roles for shep/sup-26 orthologs in the human nervous system are discussed.

Liang, C., Wang, Y., Murota, Y., Liu, X., Smith, D., Siomi, M. C. and Liu, Q. (2015). TAF11 assembles the RISC loading complex to enhance RNAi efficiency. Mol Cell 59: 807-818. PubMed ID: 26257286
Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains the Dicer-2 (Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila. However, the precise composition and action mechanism of Drosophila RLC remain unclear. This study identified the missing factor of RLC as TATA-binding protein-associated factor 11 (TAF11) by genetic screen. Although it is an annotated nuclear transcription factor, TAF11 also associated with Dcr-2/R2D2 and localizes to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11-/- ovary extract, the RLC was reconstituted in vitro using the recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, this study showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, these genetic and biochemical studies define the molecular nature of the Drosophila RLC and elucidate a cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency.

Thursday, September 10th

Schulz, K.N., Bondra, E.R., Moshe, A., Villalta, J.E., Lieb, J.D., Kaplan, T., McKay, D.J. and Harrison, M.M. (2015). Zelda is differentially required for chromatin accessibility, transcription-factor binding and gene expression in the early Drosophila embryo. Genome Res [Epub ahead of print]. PubMed ID: 26335634
The transition from a specified germ cell to a population of pluripotent cells occurs rapidly following fertilization. During this developmental transition, the zygotic genome is largely transcriptionally quiescent and undergoes significant chromatin remodeling. In Drosophila, the DNA-binding protein Zelda (also known as Vielfaltig) is required for this transition and for transcriptional activation of the zygotic genome. Open chromatin is associated with Zelda-bound loci as well as more generally with regions of active transcription. Nonetheless, the extent to which Zelda influences chromatin accessibility across the genome is largely unknown. This study used Formaldehyde Assisted Isolation of Regulatory Elements to determine the role of Zelda in regulating regions of open chromatin in the early embryo. Zelda was found to be essential for hundreds of regions of open chromatin. This Zelda-mediated chromatin accessibility facilitates transcription-factor recruitment and early gene expression. Thus, Zelda possesses some key characteristics of a pioneer factor. Unexpectedly, chromatin at a large subset of Zelda-bound regions remains open even in the absence of Zelda. The GAGA factor-binding motif and embryonic GAGA factor binding are specifically enriched in these regions. The study proposes that both Zelda and GAGA factor function to specify sites of open chromatin and together facilitate the remodeling of the early embryonic genome.

Lin, S., Ewen-Campen, B., Ni, X., Housden, B. E. and Perrimon, N. (2015). In vivo transcriptional activation using CRISPR-Cas9 in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 26245833
A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be over-expressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. This study characterized the dCas9-VPR system in Drosophila cells and in vivo. This system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and a single guide RNA upstream of the transcription start site can activate high levels of target transcription. Marked heterogeneity in guide RNA efficacy was observed for any given gene, and transcription was observed to be inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, dCas9-VPR was used to identify target genes for Twist and Snail. In addition, both Twist and Snail were simultaneously activated to identify synergistic responses to this physiologically relevant combination. Finally, dCas9-VPR were shown to activate target genes and cause dominant phenotypes in vivo. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of over-expression studies.

Bothma, J. P., Garcia, H. G., Ng, S., Perry, M. W., Gregor, T. and Levine, M. (2015). Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo. Elife 4. PubMed ID: 26267217
Metazoan genes are embedded in a rich milieu of regulatory information that often includes multiple enhancers possessing overlapping activities. This study employed quantitative live imaging methods to assess the function of pairs of primary and shadow enhancers in the regulation of key patterning genes - knirps, hunchback, and snail - in developing Drosophila embryos. The knirps enhancers exhibit additive, sometimes even super-additive activities, consistent with classical gene fusion studies. In contrast, the hunchback enhancers function sub-additively in anterior regions containing saturating levels of the Bicoid activator, but function additively in regions where there are diminishing levels of the Bicoid gradient. Strikingly sub-additive behavior is also observed for snail, whereby removal of the proximal enhancer causes a significant increase in gene expression. Quantitative modeling of enhancer-promoter interactions suggests that weakly active enhancers function additively while strong enhancers behave sub-additively due to competition with the target promoter (Bothma, 2015).

Sun, Y., Nien, C.Y., Chen, K., Liu, H.Y., Johnston, J., Zeitlinger, J. and Rushlow, C. (2015). Zelda overcomes the high intrinsic nucleosome barrier at enhancers during Drosophila zygotic genome activation. Genome Res [Epub ahead of print]. PubMed ID: 26335633
The Drosophila genome activator Vielfaltig (Vfl), also known as Zelda (Zld), is thought to prime enhancers for activation by patterning transcription factors (TFs). Such priming is accompanied by increased chromatin accessibility but the mechanisms by which this occurs are poorly understood. This study analyzes the effect of Zld on genome-wide nucleosome occupancy and binding of the patterning TF Dorsal (Dl). Early enhancers were shown to be characterized by an intrinsically high nucleosome barrier. Zld tackles this nucleosome barrier through local depletion of nucleosomes with the effect being dependent on the number and position of Zld motifs. Without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. The study proposes that Zld primes enhancers by lowering the high nucleosome barrier just enough to assist TFs in accessing their binding motifs and promoting spatially controlled enhancer activation if the right patterning TFs are present. Also, that genome activators in general will utilize this mechanism to activate the zygotic genome in a robust and precise manner.

Wednesday, September 9th

Weir, P.T. and Dickinson, M.H. (2015). Functional divisions for visual processing in the central brain of flying Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26324910
Although anatomy is often the first step in assigning functions to neural structures, it is not always clear whether architecturally distinct regions of the brain correspond to operational units. Whereas neuroarchitecture remains relatively static, functional connectivity may change almost instantaneously according to behavioral context. This study imaged panneuronal responses to visual stimuli in a highly conserved central brain region in the fruit fly, Drosophila, during flight. In one substructure, the fan-shaped body, automated analysis reveals three layers that are unresponsive in quiescent flies but become responsive to visual stimuli when the animal is flying. The responses of these regions to a broad suite of visual stimuli suggest that they are involved in the regulation of flight heading. To identify the cell types that underlie these responses, activity was imaged in sets of genetically defined neurons with arborizations in the targeted layers. The responses of this collection during flight also segregate into three sets, confirming the existence of three layers, and they collectively account for the panneuronal activity. These results provide an atlas of flight-gated visual responses in a central brain circuit.

Huckesfeld, S., Schoofs, A., Schlegel, P., Miroschnikow, A. and Pankratz, M. J. (2015). Localization of motor neurons and central pattern generators for motor patterns underlying feeding behavior in Drosophila larvae. PLoS One 10: e0135011. PubMed ID: 26252658
This study has begun to deconstruct the motor system driving Drosophila larval feeding behavior into its component parts. Distinct clusters of motor neurons were identified that execute head tilting, mouth hook movements, and pharyngeal pumping during larval feeding. This basic anatomical scaffold enabled the use of calcium-imaging to monitor the neural activity of motor neurons within the central nervous system (CNS) that drive food intake. Simultaneous nerve- and muscle-recordings demonstrate that the motor neurons innervate the cibarial dilator musculature (CDM) ipsi- and contra-laterally. By classical lesion experiments a set of CPGs generating the neuronal pattern underlying feeding movements was localized to the subesophageal zone (SEZ). Lesioning of higher brain centers decelerated all feeding-related motor patterns, whereas lesioning of ventral nerve cord (VNC) only affected the motor rhythm underlying pharyngeal pumping. These findings provide a basis for progressing upstream of the motor neurons to identify higher regulatory components of the feeding motor system.

Del Pino, F., Jara, C., Pino, L., Medina-Muñoz, M.C., Alvarez, E. and Godoy-Herrera, R. (2015). The identification of congeners and aliens by Drosophila larvae. PLoS One 10: e0136363. PubMed ID: 26313007
This study investigated the role of Drosophila larva olfactory system in identification of congeners and aliens. These activities are important in larva navigation across substrates, and have implications for allocation of space and food among species of similar ecologies. Wild type larvae of cosmopolitan D. melanogaster and endemic D. pavani, which cohabit the same breeding sites, use species-specific volatiles to identify conspecifics and aliens moving toward larvae of their species. D. gaucha larvae, a sibling species of D. pavani that is ecologically isolated from D. melanogaster, does not respond to melanogaster odor cues. Similar to D. pavani larvae, the navigation of pavani female x gaucha male hybrids is influenced by conspecific and alien odors, whereas gaucha female x pavani male hybrid larvae exhibit behavior similar to the D. gaucha parent. The two sibling species exhibit substantial evolutionary divergence in processing the odor inputs necessary to identify conspecifics. Orco (Or83b) mutant larvae of D. melanogaster, which exhibit a loss of sense of smell, do not distinguish conspecific from alien larvae, instead moving across the substrate. Syn97CS and rut larvae of D. melanogaster, which are unable to learn but can smell, move across the substrate as well. The Orco (Or83b), Syn97CS and rut loci are necessary to orient navigation by D. melanogaster larvae. Individuals of the Trana strain of D. melanogaster do not respond to conspecific and alien larval volatiles and therefore navigate randomly across the substrate. By contrast, larvae of the Til-Til strain uses larval volatiles to orient their movement. Natural populations of D. melanogaster may exhibit differences in identification of conspecific and alien larvae. Larval locomotion is not affected by the volatiles. 

Hunt, V.L., Zhong, W., McClure, C.D., Mlynski, D.T., Duxbury, E.M., Charnley, A.K. and Priest, N.K. (2015). Cold-seeking behaviour mitigates reproductive losses from fungal infection in Drosophila. J Anim Ecol [Epub ahead of print]. PubMed ID: 26332860
This study investigated the temperature preference of the fruit fly, Drosophila melanogaster, during infection with the fungal pathogen, Metarhizium robertsii, and the consequences of temperature preference on life history traits. Multiple measurements were used to assess the fitness consequences of temperature including age-specific changes in mortality rate and reproduction, providing more sensitive measures of accounting for variation in fitness with age. Flies were found to optimize their life history by exploiting thermal variation. Fungus-infected flies seek out cooler temperatures, which immediately reduces their fecundity but, ultimately, increases their lifetime reproductive success. Colder temperatures reduce fungal growth rates both in vivo and in vitro, indicating that cooler temperatures increase resistance to the infection. By comparing life history responses in infected and control animals, it was found that cold seeking in infected animals facilitates a trade off between early- and late-age reproduction, but does not otherwise provide life history benefits that are specific to infected animals. These results indicate that cold-seeking is a mechanism for reducing the reproductive costs of infection. In contrast, uninfected control flies prefer warmer temperatures that optimise reproductive success via a rapid propagation strategy. These findings help explain how life history trade-offs are mediated and how animals cope with infection, which will be increasingly important given the recent emergence of fungal pathogens and global climate change.

Zalucki, O., Day, R., Kottler, B., Karunanithi, S. and van Swinderen, B. (2015). Behavioral and electrophysiological analysis of general anesthesia in 3 background strains of Drosophila melanogaster. Fly (Austin) 9: 7-15. PubMed ID: 26267354
General anesthetics achieve behavioral unresponsiveness via a mechanism that is incompletely understood. The study of genetic model systems such as the fruit fly Drosophila melanogaster is crucial to advancing understanding of how anesthetic drugs render animals unresponsive. Previous studies have shown that wild-type control strains differ significantly in their sensitivity to general anesthetics, which potentially introduces confounding factors for comparing genetic mutations placed on these wild-type backgrounds. This study examined a variety of behavioral and electrophysiological endpoints in Drosophila, in both adult and larval animals. These endpoints where characterized in 3 commonly used fly strains: wild-type Canton Special (CS), and 2 commonly used white-eyed strains, isoCJ1 and w1118. CS and isoCJ1 were found to show remarkably similar sensitivity to isoflurane across a variety of behavioral and electrophysiological endpoints. In contrast, w1118 is resistant to isoflurane compared to the other 2 strains at both the adult and larval stages. This resistance is however not reflected at the level of neurotransmitter release at the larval neuromuscular junction (NMJ). This suggests that the w1118 strain harbors another mutation that produces isoflurane resistance, by acting on an arousal pathway that is most likely preserved between larval and adult brains. This mutation probably also affects sleep, as marked differences between isoCJ1 and ww1118 have also recently been found for behavioral responsiveness and sleep intensity measures.

Slepian, Z., Sundby, K., Glier, S., McDaniels, J., Nystrom, T., Mukherjee, S., Acton, S. T. and Condron, B. (2015). Visual attraction in Drosophila larvae develops during a critical period and is modulated by crowding conditions. J Comp Physiol A Neuroethol Sens Neural Behav Physiol [Epub ahead of print]. PubMed ID: 26265464
The development of social behavior is poorly understood. Many animals adjust their behavior to environmental conditions based on a social context. Despite having relatively simple visual systems, Drosophila larvae are capable of identifying and are attracted to the movements of other larvae. This study shows that Drosophila larval visual recognition is encoded by the movements of nearby larvae, experienced during a specific developmental critical period. Exposure to moving larvae, only during a specific period, is sufficient for later visual recognition of movement. Larvae exposed to wild-type body movements, during the critical period, are not attracted to the movements of tubby mutants, which have altered morphology. However, exposure to tubby, during the critical period, results in tubby recognition at the expense of wild-type recognition indicating that this is true learning. Visual recognition is not learned in excessively crowded conditions, and this is emulated by exposure, during the critical period, to food previously used by crowded larvae. It is propose that Drosophila larvae have a distinct critical period, during which they assess both social and resource conditions, and that this irreversibly determines later visually guided social behavior. This model provides a platform towards understanding the regulation and development of social behavior.

Tuesday, September 8th

Xu, S. and Elefant, F. (2015). Tip off the HAT- Epigenetic control of learning and memory by Drosophila Tip60. Fly (Austin) 9: 22-28. PubMed ID: 26327426
Disruption of epigenetic gene control mechanisms involving histone acetylation in the brain causes cognitive impairment, a debilitating hallmark of most neurodegenerative disorders. Histone acetylation regulates cognitive gene expression via chromatin packaging control in neurons. Unfortunately, the histone acetyltransferases (HATs) that generate such neural epigenetic signatures and their mechanisms of action remain unclear. Recent findings provide insight into this question by demonstrating that Tip60 HAT action is critical for morphology and function of the mushroom body (MB), the learning and memory center in the Drosophila brain. This study shows that Tip60 is robustly produced in MB Kenyon cells and extending axonal lobes and that target MB Tip60 HAT loss results in axonal outgrowth disruption. Functional consequences of loss and gain of Tip60 HAT levels in the MB are evidenced by defects in memory. Tip60 ChIP-Seq analysis reveals enrichment for genes that function in cognitive processes and accordingly, key genes representing these pathways are misregulated in the Tip60 HAT mutant fly brain. Remarkably, increasing levels of Tip60 in the MB rescues learning and memory deficits resulting from Alzheimer's disease associated amyloid precursor protein (APP) induced neurodegeneration. These data highlight the potential of HAT activators as a therapeutic option for cognitive disorders.

Ea, V., Sexton, T., Gostan, T., Herviou, L., Baudement, M. O., Zhang, Y., Berlivet, S., Le Lay-Taha, M. N., Cathala, G., Lesne, A., Victor, J. M., Fan, Y., Cavalli, G. and Forne, T. (2015). Distinct polymer physics principles govern chromatin dynamics in mouse and Drosophila topological domains. BMC Genomics 16: 607. PubMed ID: 26271925
In higher eukaryotes, the genome is partitioned into large "Topologically Associating Domains" (TADs) in which the chromatin displays favoured long-range contacts. This study used data from diverse 3C-derived methods to explore chromatin dynamics within mouse and Drosophila TADs. In mouse Embryonic Stem Cells (mESC), that possess large TADs (median size of 840 kb), it was shown that the statistical helix model, but not globule models, is relevant not only in gene-rich TADs, but also in gene-poor and gene-desert TADs. Interestingly, this statistical helix organization is considerably relaxed in mESC compared to liver cells, indicating that the impact of the constraints responsible for this organization is weaker in pluripotent cells. Finally, depletion of histone H1 in mESC alters local chromatin flexibility but not the statistical helix organization. In Drosophila, which possesses TADs of smaller sizes (median size of 70 kb), while chromatin compaction and flexibility are finely tuned according to the epigenetic landscape, chromatin dynamics within TADs is generally compatible with an unconstrained polymer configuration. It is concluded that models issued from polymer physics can accurately describe the organization principles governing chromatin dynamics in both mouse and Drosophila TADs.

Singh, N. P. and Mishra, R. K. (2015). Specific combinations of boundary element and Polycomb response element are required for the regulation of the Hox genes in Drosophila melanogaster. Mech Dev [Epub ahead of print]. PubMed ID: 26254901
In the bithorax complex of Drosophila melanogaster, the chromatin boundary elements (BE) demarcate cis-regulatory domains that regulate Hox genes along the anteroposterior body axis. These elements are closely associated with the Polycomb Response Elements (PREs) and restrict the ectopic activation of cis-regulatory domains during development. The relevance of such specific genomic arrangements of regulatory elements remains unclear. Deletions of individual BE-PRE combination result in distinct homeotic phenotypes. This study shows that deletion of two such BE-PRE combinations in cis leads to new genetic interactions, which manifests as dorsal closure defect phenotype in adult abdominal epithelia. The dorsal closure phenotype results from enhanced and ectopic expression of Hox gene Abd-B in the larval epithelial cells. This suggests a specific role of multiple BE-PRE combinations in the larval epithelial cells for regulation of Abd-B. Using chromosome conformation capture experiments, this study shows that genetic interactions correlate with direct physical interactions among the BE-PRE combinations. These results demonstrate the functional relevance of the closely associated BE and PRE combinations in regulation of Hox genes.

Garavís, M., Méndez-Lago, M., Gabelica, V., Whitehead, S.L., González, C. and Villasante, A. (2015). The structure of an endogenous Drosophila centromere reveals the prevalence of tandemly repeated sequences able to form i-motifs. Sci Rep 5: 13307. PubMed ID: 26289671
Centromeres are the chromosomal loci at which spindle microtubules attach to mediate chromosome segregation during mitosis and meiosis. In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements). Despite the efforts to establish complete genomic sequences of eukaryotic organisms, the so-called 'finished' genomes are not actually complete because the centromeres have not been assembled due to the intrinsic difficulties in constructing both physical maps and complete sequence assemblies of long stretches of tandemly repetitive DNA. This study shows the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. a four-stranded intercalated structure formed by the association of two parallel duplexes combined in an antiparallel fashion. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads to suggestion that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures. 

Monday, September 7th

Gailite, I., Aerne, B. L. and Tapon, N. (2015). Differential control of Yorkie activity by LKB1/AMPK and the Hippo/Warts cascade in the central nervous system. Proc Natl Acad Sci [Epub ahead of print] PubMed ID: 26324895
The Hippo (Hpo) pathway is a highly conserved tumor suppressor network that restricts developmental tissue growth and regulates stem cell proliferation and differentiation. At the heart of the Hpo pathway is the progrowth transcriptional coactivator Yorkie. Yki activity is restricted through phosphorylation by the Hpo/Warts core kinase cascade, but increasing evidence indicates that core kinase-independent modes of regulation also play an important role. This study examined Yki regulation in the Drosophila larval CNS and uncovered a Hpo/Warts-independent function for the tumor suppressor kinase LKB1 and its downstream effector, the energy sensor AMP-activated protein kinase (AMPK), in repressing Yki activity in the central brain. Thus Yki is inhibited by the nutrient-sensing LKB1/AMPK cascade independent of Hpo/ Warts in a population of neural progenitors in the developing Drosophila larval brain. Although the Hpo/Warts core cascade restrains Yki in the optic lobe, it is dispensable for Yki target gene repression in the late larval central brain. Thus, this study demonstrates a dramatically different wiring of Hpo signaling in neighboring cell populations of distinct developmental origins in the central nervous system.

Patel, P. H., Dutta, D. and Edgar, B. A. (2015). Niche appropriation by Drosophila intestinal stem cell tumours. Nat Cell Biol 17: 1182-1192. PubMed ID: 26237646
Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. This study investigated Drosophila intestinal stem cell (ISC) tumours generated by suppressing Notch (N) signalling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumour initiation and an autocrine EGFR ligand, Spitz, during early tumour growth. On achieving a critical mass these tumours displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumour growth without the need for secondary mutations in growth signalling pathways. The appropriation of niche signalling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis.

Mitchell, N. C., Tchoubrieva, E. B., Chahal, A., Woods, S., Lee, A., Lin, J. I., Parsons, L., Jastrzebski, K., Poortinga, G., Hannan, K. M., Pearson, R. B., Hannan, R. D. and Quinn, L. M. (2015). S6 kinase is essential for MYC-dependent rDNA transcription in Drosophila. Cell Signal 27: 2045-2053. PubMed ID: 26215099
Increased rates of ribosome biogenesis and biomass accumulation are fundamental properties of rapidly growing and dividing malignant cells. The MYC oncoprotein drives growth predominantly via its ability to upregulate the ribosome biogenesis program, in particular stimulating the activity of the RNA Polymerase I (Pol I) machinery to increase ribosomal RNA (rRNA) transcription. Although MYC function is known to be highly dependent on the cellular signalling context, the pathways interacting with MYC to regulate transcription of ribosomal genes (rDNA) in vivo in response to growth factor status, nutrient availability and cellular stress are only beginning to be understood. To determine factors critical to MYC-dependent stimulation of rDNA transcription in vivo, a transient expression screen for known oncogenic signalling pathways was performed in Drosophila. Strikingly, from the broad range of pathways tested, ribosomal protein S6 Kinase (S6K) activity, downstream of the TOR pathway, was the only factor rate-limiting for the rapid induction of rDNA transcription due to transiently increased MYC. Further, one of the mechanism(s) by which MYC and S6K cooperate was shown to be through coordinate activation of the essential Pol I transcription initiation factor TIF-1A (RRN 3). As Pol I targeted therapy is now in phase 1 clinical trials in patients with haematological malignancies, including those driven by MYC, these data suggest that therapies dually targeting Pol I transcription and S6K activity may be effective in treating MYC-driven tumours.

Bai, H., Post, S., Kang, P. and Tatar, M. (2015). Drosophila longevity assurance conferred by reduced Insulin receptor substrate Chico partially requires d4eBP. PLoS One 10: e0134415. PubMed ID: 26252766
Mutations of the insulin/IGF signaling (IIS) pathway extend Drosophila lifespan. Based on genetic epistasis analyses, this longevity assurance is attributed to downstream effects of the FOXO transcription factor. However, as reported FOXO accounts for only a portion of the observed longevity benefit, suggesting there are additional outputs of IIS to mediate aging. One candidate is target of rapamycin complex 1 (TORC1). Reduced TORC1 activity is reported to slow aging, whereas reduced IIS is reported to repress TORC1 activity. The eukaryotic translation initiation factor 4E binding protein (4E-BP) is repressed by TORC1, and activated 4E-BP is reported to increase Drosophila lifespan. This study use genetic epistasis analyses to test whether longevity assurance mutants of chico, the Drosophila insulin receptor substrate homolog, require Drosophila d4eBP to slow aging. In chico heterozygotes, which are robustly long-lived, d4eBP is required but not sufficient to slow aging. Remarkably, d4eBP is not required or sufficient for chico homozygotes to extend longevity. Likewise, chico heterozygote females partially require d4eBP to preserve age-dependent locomotion, and both chico genotypes require d4eBP to improve stress-resistance. Reproduction and most measures of growth affected by either chico genotype are always independent of d4eBP. In females, chico heterozygotes paradoxically produce more rather than less phosphorylated 4E-BP (p4E-BP). Altered IRS function within the IIS pathway of Drosophila appears to have partial, conditional capacity to regulate aging through an unconventional interaction with 4E-BP.

Sunday, September 6th

Handu, M., Kaduskar, B., Ravindranathan, R., Soory, A., Giri, R., Elango, V.B., Gowda, H. and Ratnaparkhi, G.S. (2015). SUMO enriched proteome for Drosophila innate immune response. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26290570
SUMO modification modulates the expression of defense genes in Drosophila, activated by the Toll/NF-κB and IMD/NF-κB signaling networks. There is however limited understanding of the SUMO modulated regulation of the immune response and lack of information on SUMO targets in the immune system. This study measured the changes to the SUMO proteome in S2 cells in response to an LPS challenge, identifying 1619 unique proteins in SUMO enriched lysates. A confident set of 710 proteins represents the immune induced SUMO proteome. A subset of the confident set were validated and shown to be bona-fide SUMO targets. These include components of immune signaling pathways such as Caspar, Jra, Kay, cdc42, p38b, 14-3-3ϵ, as also cellular proteins with diverse functions, many being components of protein complexes, such as prosβ4, Rps10b, SmD3, Tango7 and Aats-arg. This study is one of the first to describe SUMO proteome for the Drosophila immune response. These data and analysis provide a global framework for the understanding of SUMO modification in the host response to pathogens.

Merkling, S. H., Overheul, G. J., van Mierlo, J. T., Arends, D., Gilissen, C. and van Rij, R. P. (2015). The heat shock response restricts virus infection in Drosophila. Sci Rep 5: 12758. PubMed ID: 26234525
Innate immunity is the first line of defence against pathogens and is essential for survival of the infected host. Drosophila is an emerging model to study viral pathogenesis, yet antiviral defence responses remain poorly understood. This study describes the heat shock response, a cellular mechanism that prevents proteotoxicity, as a component of the antiviral immune response in Drosophila. Transcriptome analyses of Drosophila S2 cells and adult flies revealed strong induction of the heat shock response upon RNA virus infection. Dynamic induction patterns of heat shock pathway components were characterized in vitro and in vivo following infection with different classes of viruses. The heat shock transcription factor (Hsf), as well as active viral replication, were necessary for the induction of the response. Hsf-deficient adult flies were hypersensitive to virus infection, indicating a role of the heat shock response in antiviral defence. In accordance, transgenic activation of the heat shock response prolonged survival time after infection and enabled long-term control of virus replication to undetectable levels. Together, these results establish the heat shock response as an important constituent of innate antiviral immunity in Drosophila.

Bou Sleiman, M. S., Osman, D., Massouras, A., Hoffmann, A. A., Lemaitre, B. and Deplancke, B. (2015). Genetic, molecular and physiological basis of variation in Drosophila gut immunocompetence. Nat Commun 6: 7829. PubMed ID: 26213329
Gut immunocompetence involves immune, stress and regenerative processes. To investigate the determinants underlying inter-individual variation in gut immunocompetence, enteric infection was performed of 140 Drosophila lines with the entomopathogenic bacterium Pseudomonas entomophila, and extensive variation was observed in survival. Using genome-wide association analysis, several novel immune modulators were identified. Transcriptional profiling further shows that the intestinal molecular state differs between resistant and susceptible lines, already before infection, with one transcriptional module involving genes linked to reactive oxygen species (ROS) metabolism contributing to this difference. This genetic and molecular variation is physiologically manifested in lower ROS activity, lower susceptibility to ROS-inducing agent, faster pathogen clearance and higher stem cell activity in resistant versus susceptible lines. This study revealed how relatively minor, but systematic variation can mediate overt physiological differences that determine enteric infection susceptibility.

Longdon, B., Hadfield, J. D., Day, J. P., Smith, S. C., McGonigle, J. E., Cogni, R., Cao, C. and Jiggins, F. M. (2015). The causes and consequences of changes in virulence following pathogen host shifts. PLoS Pathog 11: e1004728. PubMed ID: 25774803
Emerging infectious diseases are often the result of a host shift, where the pathogen originates from a different host species. Virulence--the harm a pathogen does to its host-can be extremely high following a host shift (for example Ebola, HIV, and SARs), while other host shifts may go undetected as they cause few symptoms in the new host. This study examined how virulence varies across host species by carrying out a large cross infection experiment using 48 species of Drosophilidae and an RNA virus. Host shifts resulted in dramatic variation in virulence, with benign infections in some species and rapid death in others. The change in virulence was highly predictable from the host phylogeny, with hosts clustering together in distinct clades displaying high or low virulence. High levels of virulence are associated with high viral loads, and this may determine the transmission rate of the virus.

Saturday, September 5th

Bruckert, H., Marchetti, G., Ramialison, M. and Besse, F. (2015). Drosophila Hrp48 is required for mushroom body axon growth, branching and guidance. PLoS One 10: e0136610. PubMed ID: 26313745
RNA binding proteins assemble on mRNAs to control every single step of their life cycle, from nuclear splicing to cytoplasmic localization, stabilization or translation. This study investigated the role of Drosophila Hrp48, a fly homologue of mammalian hnRNP A2/B1, during central nervous system development. Using a combination of mutant conditions, hrp48 was shown to be required for the formation, growth and guidance of axonal branches in Mushroom Body neurons. Furthermore, hrp48 inactivation induces an overextension of Mushroom Body dorsal axonal branches, with a significantly higher penetrance in females than in males. Finally, as demonstrated by immunolocalization studies, Hrp48 is confined to Mushroom Body neuron cell bodies, where it accumulates in the cytoplasm from larval stages to adulthood. Altogether, these data provide evidence for a crucial in vivo role of the hnRNP Hrp48 in multiple aspects of axon guidance and branching during nervous system development. They also indicate cryptic sex differences in the development of sexually non-dimorphic neuronal structures.

Petsakou, A., Sapsis, T. P. and Blau, J. (2015). Circadian rhythms in Rho1 activity regulate neuronal plasticity and network hierarchy. Cell 162: 823-835. PubMed ID: 26234154
Neuronal plasticity helps animals learn from their environment. However, it is challenging to link specific changes in defined neurons to altered behavior. This study focused on circadian rhythms in the structure of the principal s-LNv clock neurons in Drosophila. By quantifying neuronal architecture, s-LNv structural plasticity was found to change the amount of axonal material in addition to cycles of fasciculation and defasciculation. This is controlled by rhythmic Rho1 activity that retracts s-LNv axonal termini by increasing myosin phosphorylation and simultaneously changes the balance of pre-synaptic and dendritic markers. This plasticity is required to change clock network hierarchy and allow seasonal adaptation. Rhythms in Rho1 activity are controlled by clock-regulated transcription of Puratrophin-1-like (Pura), a Rho1 GEF. Since spinocerebellar ataxia is associated with mutations in human Puratrophin-1, these data support the idea that defective actin-related plasticity underlies this ataxia.

Adewoye, A. B., Kyriacou, C. P. and Tauber, E. (2015). Identification and functional analysis of early gene expression induced by circadian light-resetting in Drosophila. BMC Genomics 16: 570. PubMed ID: 26231660
The environmental light-dark cycle is the dominant cue that maintains 24-h biological rhythms in multicellular organisms. In Drosophila, light entrainment is mediated by the photosensitive protein Cryptochrome, but the role and extent of transcription regulation in light resetting of the dipteran clock is yet unknown. Given the broad transcriptional changes in response to light previously identified in mammals, this study sought to analyse light-induced global transcriptional changes in the fly's head by using Affymetrix microarrays. Flies were subjected to a 30-min light pulse during the early night (3 h after lights-off), a stimulus which causes a substantial phase delay of the circadian rhythm. Changes were then analyzed in gene expression 1 h after the light stimulus. Two hundred genes were identified whose transcripts were significantly altered in response to the light pulse at a false discovery rate cut-off of 10%. Analysis of these genes and their biological functions suggests the involvement of at least six biological processes in light-induced delay phase shifts of rhythmic activities. These processes include signalling, ion channel transport, receptor activity, synaptic organisation, signal transduction, and chromatin remodelling. Using RNAi, the expression of 22 genes was downregulated in the clock neurons, leading to significant effects on circadian output. For example, while continuous light normally causes arrhythmicity in wild-type flies, the knockdown of Kr-h1, Nipped-A, Thor, nrv1, Nf1, CG11155 (ionotropic glutamate receptor), and Fmr1 resulted in flies that were rhythmic, suggesting a disruption in the light input pathway to the clock. This analysis provides a first insight into the early responsive genes that are activated by light and their contribution to light resetting of the Drosophila clock. The analysis suggests multiple domains and pathways that might be associated with light entrainment, including a mechanism that was represented by a light-activated set of chromatin remodelling genes.

Gresser, A. L., Gutzwiller, L. M., Gauck, M. K., Hartenstein, V., Cook, T. A. and Gebelein, B. (2015). Rhomboid enhancer activity defines a subset of Drosophila neural precursors required for proper feeding, growth and viability. PLoS One 10: e0134915. PubMed ID: 26252385
Organismal growth regulation requires the interaction of multiple metabolic, hormonal and neuronal pathways. While the molecular basis for many of these are well characterized, less is known about the developmental origins of growth regulatory structures and the mechanisms governing control of feeding and satiety. For these reasons, new tools and approaches are needed to link the specification and maturation of discrete cell populations with their subsequent regulatory roles. This study characterized a rhomboid enhancer element that selectively labels four Drosophila embryonic neural precursors. These precursors give rise to the hypopharyngeal sensory organ of the peripheral nervous system and a subset of neurons in the deutocerebral region of the embryonic central nervous system. Post embryogenesis, the rhomboid enhancer is active in a subset of cells within the larval pharyngeal epithelium. Enhancer-targeted toxin expression alters the morphology of the sense organ and results in impaired larval growth, developmental delay, defective anterior spiracle eversion and lethality. Limiting the duration of toxin expression reveals differences in the critical periods for these effects. Embryonic expression causes developmental defects and partially penetrant pre-pupal lethality. Survivors of embryonic expression, however, ultimately become viable adults. In contrast, post-embryonic toxin expression results in fully penetrant lethality. To better define the larval growth defect, a variety of assays were used to demonstrate that toxin-targeted larvae are capable of locating, ingesting and clearing food and they exhibit normal food search behaviors. Strikingly, however, following food exposure these larvae show a rapid decrease in consumption suggesting a satiety-like phenomenon that correlates with the period of impaired larval growth. Together, these data suggest a critical role for these enhancer-defined lineages in regulating feeding, growth and viability.

Friday, September 4th

Gottardo, M., Pollarolo, G., Llamazares, S., Reina, J., Riparbelli, M.G., Callaini, G. and Gonzalez, C. (2015). Loss of Centrobin enables daughter centrioles to form sensory cilia in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 26299513
Sensory cilia are organelles that convey information to the cell from the extracellular environment. In vertebrates, ciliary dysfunction results in ciliopathies that in humans comprise a wide spectrum of developmental disorders. In Drosophila, sensory cilia are found only in the neurons of type I sensory organs; ciliary dysfunction impairs the mechanosensory properties of bristles and chaetae and leads to uncoordination. The cilium is defined by the ciliary membrane, a protrusion of the cell membrane that envelops the core structure known as the axoneme, a microtubule array that extends along the cilium from the basal body. In vertebrates, basal body function requires centriolar distal and subdistal appendages and satellites. Because these structures are acquired through centriole maturation, only mother centrioles can serve as basal bodies. This study shows that although centriole maturity traits are lacking in Drosophila, basal body fate is reserved to mother centrioles in Drosophila type I neurons. Moreover, it was shown that depletion of the daughter-centriole-specific protein Centrobin (CNB) enables daughter centrioles to dock on the cell membrane and to template an ectopic axoneme that, although structurally defective, protrudes out of the cell and is enveloped by a ciliary membrane. Conversely, basal body capability is inhibited in mother centrioles modified to carry CNB. These results reveal the crucial role of CNB in regulating basal body function in Drosophila ciliated sensory organs.

Hunter, M.V., Lee, D.M., Harris, T.J. and Fernandez-Gonzalez, R. (2015). Polarized E-cadherin endocytosis directs actomyosin remodeling during embryonic wound repair. J Cell Biol [Epub ahead of print]. PubMed ID: 26304727
Embryonic epithelia have a remarkable ability to rapidly repair wounds. A supracellular actomyosin cable around the wound coordinates cellular movements and promotes wound closure. Actomyosin cable formation is accompanied by junctional rearrangements at the wound margin. This study used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility. Blocking endocytosis with pharmacological or genetic approaches disrupts wound repair. The defect in wound closure is accompanied by impaired removal of E-cadherin from the wound edge and defective actomyosin cable assembly. E-cadherin overexpression also results in reduced actin accumulation around wounds and slower wound closure. Reducing E-cadherin levels in embryos in which endocytosis is blocked rescues actin localization to the wound margin. These results demonstrate a central role for endocytosis in wound healing and indicate that polarized E-cadherin endocytosis is necessary for actomyosin remodeling during embryonic wound repair.

Munjal, A., Philippe, J. M., Munro, E. and Lecuit, T. (2015). A self-organized biomechanical network drives shape changes during tissue morphogenesis. Nature 524: 351-355. PubMed ID: 26214737
Tissue morphogenesis is orchestrated by cell shape changes. Forces required to power these changes are generated by non-muscle myosin II (MyoII) motor proteins pulling filamentous actin (F-actin). Actomyosin networks undergo cycles of assembly and disassembly (pulses) to cause cell deformations alternating with steps of stabilization to result in irreversible shape changes. Although this ratchet-like behaviour operates in a variety of contexts, the underlying mechanisms remain unclear. This study investigated the role of MyoII regulation through the conserved Rho1-Rok pathway during Drosophila melanogaster germband extension. This morphogenetic process is powered by cell intercalation, which involves the shrinkage of junctions in the dorsal-ventral axis (vertical junctions) followed by junction extension in the anterior-posterior axis. While polarized flows of medial-apical MyoII pulses deform vertical junctions, MyoII enrichment on these junctions (planar polarity) stabilizes them. Two critical properties of MyoII dynamics were identified that underlie stability and pulsatility: exchange kinetics governed by phosphorylation-dephosphorylation cycles of the MyoII regulatory light chain; and advection due to contraction of the motors on F-actin networks. Spatial control over MyoII exchange kinetics establishes two stable regimes of high and low dissociation rates, resulting in MyoII planar polarity. Pulsatility emerges at intermediate dissociation rates, enabling convergent advection of MyoII and its upstream regulators Rho1 GTP, Rok and MyoII phosphatase. Notably, pulsatility is not an outcome of an upstream Rho1 pacemaker. Rather, it is a self-organized system that involves positive and negative biomechanical feedback between MyoII advection and dissociation rates.

Myat, M.M., Rashmi, R.N., Manna, D., Xu, N., Patel, U., Galiano, M., Zielinski, K., Lam, A. and Welte, M.A. (2015). Drosophila KASH-domain protein Klarsicht regulates microtubule stability and integrin receptor localization during collective cell migration. Dev Biol [Epub ahead of print]. PubMed ID: 26247519
During collective migration of the Drosophila embryonic salivary gland, cells rearrange to form a tube of a distinct shape and size. This study reports a novel role for the Drosophila Klarsicht-Anc-Syne Homology (KASH) domain protein Klarsicht (Klar) in the regulation of microtubule (MT) stability and integrin receptor localization during salivary gland migration. In wild-type salivary glands, MTs become progressively stabilized as gland migration progresses. In embryos specifically lacking the KASH domain containing isoforms of Klar, salivary gland cells fail to rearrange and migrate, and these defects are accompanied by decreased MT stability and altered integrin receptor localization. In muscles and photoreceptors, KASH isoforms of Klar work together with Klaroid (Koi), a SUN domain protein, to position nuclei; however, loss of Koi has no effect on salivary gland migration, suggesting that Klar controls gland migration through novel interactors. The disrupted cell rearrangement and integrin localization observed in klar mutants could be mimicked by overexpressing Spastin (Spas), a MT severing protein, in otherwise wild-type salivary glands. In turn, promoting MT stability by reducing spas gene dosage in klar mutant embryos rescues the integrin localization, cell rearrangement and gland migration defects. Klar genetically interacts with the Rho1 small GTPase in salivary gland migration and is required for the subcellular localization of Rho1. It was also shown that Klar binds tubulin directly in vitro. These results provide the first evidence that a KASH-domain protein regulates the MT cytoskeleton and integrin localization during collective cell migration.

Thursday, September 3rd

Malik, B. R., Godena, V. K. and Whitworth, A. J. (2015). VPS35 pathogenic mutations confer no dominant toxicity but partial loss of function in Drosophila and genetically interact with parkin. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26251041
Mutations in VPS35 (PARK17) cause autosomal dominant, late onset Parkinson's disease (PD). VPS35 forms a core component of the retromer complex that mediates the retrieval of membrane proteins from endosomes back to either the Golgi or plasma membrane. While aberrant endosomal protein sorting has been linked to several neurodegenerative diseases, the mechanisms by which VPS35 mutations and retromer function contribute to PD pathogenesis are not clear. To address this, transgenic Drosophila were generated that express variant forms of human VPS35 found in PD cases and the corresponding variants of the Drosophila ortholog. No evidence was found of dominant toxicity from any variant form including the pathogenic D620N mutation, even with aging. However, assessing the ability of Vps35 variants to rescue multiple vps35-mutant phenotypes, the D620N mutation was found to confer a partial loss of function. Recently, VPS35 has been linked to the formation of mitochondria-derived vesicles, which mediate the degradation of mitochondrial proteins and contribute to mitochondrial quality control. This process is also promoted by two other PD-lined genes parkin (PARK2) and PINK1 (PARK6). This study demonstrates that vps35 genetically interacts with parkin but interestingly not with pink1. Strikingly, Vps35 overexpression is able to rescue several parkin-mutant phenotypes. Together these findings provide in vivo evidence that the D620N mutation likely confers pathogenicity through a partial loss of function mechanism and that this may be linked to other known pathogenic mechanisms such as mitochondrial dysfunction.

Zhang, S., Xie, J., Xia, Y., Yu, S., Gu, Z., Feng, R., Luo, G., Wang, D., Wang, K., Jiang, M., Cheng, X., Huang, H., Zhang, W. and Wen, T. (2015). LK6/Mnk2a is a new kinase of alpha synuclein phosphorylation mediating neurodegeneration. Sci Rep 5: 12564. PubMed ID: 26220523
Parkinson's disease (PD) is a movement disorder due to the loss of dopaminergic (DA) neurons in the substantia nigra. Alpha-synuclein phosphorylation and alpha-synuclein inclusion (Lewy body) become a main contributor, but little is known about their formation mechanism. This study used protein expression profiling of PD to construct a model of their signalling network from Drosophila to human, and major nodes were nominated that regulate PD development. LK6, a serine/threonine protein kinase, was found to play a key role in promoting alpha-synuclein Ser129 phosphorylation by identification of LK6 knockout and overexpression. In vivo tests further confirmed that LK6 indeed enhances alpha-synuclein phosphorylation, accelerates the death of dopaminergic neurons, reduces the climbing ability and shortens the the life span of Drosophila. Further, MAP kinase-interacting kinase 2a (Mnk2a), a human homolog of LK6, also been shown to make alpha-synuclein phosphorylation and leads to alpha-synuclein inclusion formation. On the mechanism, the phosphorylation mediated by LK6 and Mnk2a is controlled through ERK signal pathway by phorbolmyristate acetate (PMA) activation and PD98059 inhibition. These findings establish pivotal role of Lk6 and Mnk2a in unprecedented signalling networks, and may lead to new therapies preventing alpha-synuclein inclusion formation and neurodegeneration.

Lin, R., Rittenhouse, D., Sweeney, K., Potluri, P. and Wallace, D. C. (2015). TSPO, a mitochondrial outer membrane protein, controls ethanol-related behaviors in Drosophila. PLoS Genet 11: e1005366. PubMed ID: 26241038
The heavy consumption of ethanol can lead to alcohol use disorders (AUDs) which impact patients, their families, and societies. Yet the genetic and physiological factors that predispose humans to AUDs remain unclear. One hypothesis is that alterations in mitochondrial function modulate neuronal sensitivity to ethanol exposure. Using Drosophila genetics this study reports that inactivation of the mitochondrial outer membrane Translocator protein 18kDa (TSPO), also known as the peripheral benzodiazepine receptor, affects ethanol sedation and tolerance in male flies. Knockdown of dTSPO in adult male neurons results in increased sensitivity to ethanol sedation, and this effect requires the dTSPO depletion-mediated increase in reactive oxygen species (ROS) production and inhibition of caspase activity in fly heads. Systemic loss of dTSPO in male flies blocks the development of tolerance to repeated ethanol exposures, an effect that is not seen when dTSPO is only inactivated in neurons. Female flies are naturally more sensitive to ethanol than males, and female fly heads have strikingly lower levels of dTSPO mRNA than males. Hence, mitochondrial TSPO function plays an important role in ethanol sensitivity and tolerance. Since a large array of benzodiazepine analogues have been developed that interact with the peripheral benzodiazepine receptor, the mitochondrial TSPO might provide an important new target for treating AUDs.

Bourdet, I., Lampin-Saint-Amaux, A., Preat, T. and Goguel, V. (2015). Amyloid-β peptide exacerbates the memory deficit caused by Amyloid Precursor Protein loss-of-function in Drosophila. PLoS One 10: e0135741. PubMed ID: 26274614
The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD). APP can undergo two exclusive proteolytic pathways: cleavage by the α-secretase initiates the non-amyloidogenic pathway while cleavage by the β-secretase initiates the amyloidogenic pathway that leads, after a second cleavage by the γ-secretase, to amyloid-β (Aβ) peptides that can form toxic extracellular deposits, a hallmark of AD. The initial events leading to AD are still unknown. Importantly, aside from Aβ toxicity whose molecular mechanisms remain elusive, several studies have shown that APP plays a positive role in memory, raising the possibility that APP loss-of-function may participate to AD. APPL, the Drosophila APP ortholog, is required for associative memory in young flies. The present report provides the first analysis of the amyloidogenic pathway's influence on memory in the adult. Transient overexpression of the β-secretase, beta-site APP-cleaving enzyme, in the mushroom bodies, the center for olfactory memory, did not alter memory. In sharp contrast, β-secretase overexpression affected memory when associated with APPL partial loss-of-function. Interestingly, similar results were observed with Drosophila Aβ peptide. Because Aβ overexpression impaired memory only when combined to APPL partial loss-of-function, the data suggest that Aβ affects memory through the APPL pathway. Thus, memory is altered by two connected mechanisms-APPL loss-of-function and amyloid peptide toxicity-revealing in Drosophila a functional interaction between APPL and amyloid peptide.

Wednesday, September 2nd

Zhang, K., et al. (2015). The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature [Epub ahead of print]. PubMed ID: 26308891
The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and protein interactors for the G4C2 RNA including RanGAP1 have been previously identified. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention.

Sreedharan, J., Neukomm, L. J., Brown, R. H., Jr. and Freeman, M. R. (2015). Age-dependent TDP-43-mediated motor neuron degeneration requires GSK3, hat-trick, and xmas-2. Curr Biol 25: 2130-2136. PubMed ID: 26234214
The RNA-processing protein TDP-43 is central to the pathogenesis of amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron (MN) disease. TDP-43 is conserved in Drosophila, where it has been the topic of considerable study, but how TDP-43 mutations lead to age-dependent neurodegeneration is unclear and most approaches have not directly examined changes in MN morphology with age. This study used a mosaic approach to study age-dependent MN loss in the adult fly leg where it is possible to resolve single motor axons, NMJs and active zones, and perform rapid forward genetic screens. Expression of the mutant protein TDP-43(Q331K) caused dying-back of NMJs and axons, which could not be suppressed by mutations that block Wallerian degeneration. Three genes were identified that suppress TDP-43 toxicity, including shaggy/GSK3, a known modifier of neurodegeneration. The two additional novel suppressors, hat-trick and xmas-2, function in chromatin modeling and RNA export, two processes recently implicated in human ALS. Loss of shaggy/GSK3, hat-trick, or xmas-2 does not suppress Wallerian degeneration, arguing TDP-43(Q331K)-induced and Wallerian degeneration are genetically distinct processes. In addition to delineating genetic factors that modify TDP-43 toxicity, these results establish the Drosophila adult leg as a valuable new tool for the in vivo study of adult MN phenotypes.

Freibaum, B.D., Lu, Y., Lopez-Gonzalez, R., Kim, N.C., et al. (2015). GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature [Epub ahead of print]. PubMed ID: 26308899
The GGGGCC (G4C2) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia. The basis for pathogenesis is unknown. To elucidate the consequences of G4C2 repeat expansion in a tractable genetic system, this study generated transgenic fly lines expressing 8, 28 or 58 G4C2-repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. These transgenic animals were shown to display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, morphological abnormalities were found in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats. Moreover, a substantial defect in RNA export was identified that resulted in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.

Cheng, C. W., Lin, M. J. and Shen, C. J. (2015). Rapamycin alleviates pathogenesis of a new Drosophila model of ALS-TDP. J Neurogenet: 1-47. PubMed ID: 26219309
TDP-43 is a multi-functional RNA/DNA-binding protein well-conserved among many species including mammals and Drosophila. However, it is also a major component of the pathological inclusions associated with degenerating motor neurons of amyotrophic lateral sclerosis (ALS). Further, TDP-43 is a signature protein in one subtype of frontotemporal degeneration, FTLD-U. Currently, there are no effective drugs for these neurodegenerative diseases. This study describes the generation and characterization of a new fly model of ALS-TDP with transgenic expression of the Drosophila ortholog of TDP-43, dTDP, in adult flies under the control of a temperature sensitive motor neuron-specific GAL4, thus bypassing the deleterious effect of dTDP during development. Diminished lifespan as well as impaired locomotor activities of the flies following induction of dTDP overexpression have been observed. Dissection of the T1/T2 region of the thoracic ganglia has revealed loss of these neurons. To counter the defects in this fly model of ALS-TDP, the therapeutic effects of the autophagy activator rapamycin were examined. Although harmful to the control flies, administration of 400 muM rapamycin before the induction of dTDP overexpression can significantly reduce the number of neurons bearing dTDP + aggregates as well as partially rescue the diminished lifespan and locomotive defects of the ALS-TDP flies. Furthermore, S6K, a downstream mediator of TOR pathway, was identified as one genetic modifier of dTDP. In sum, this Drosophila model of ALS-TDP under temporal and spatial control presents a useful new genetic tool for the screening and validation of therapeutic drugs for ALS. Furthermore, the data support previous findings that autophagy activators including rapamycin are potential therapeutic drugs for the progression of neurodegenerative diseases with TDP-43 proteinopathies.

Tuesday, September 1st

Sato, K., Iwasaki, Y. W., Shibuya, A., Carninci, P., Tsuchizawa, Y., Ishizu, H., Siomi, M. C. and Siomi, H. (2015) . Krimper enforces an antisense bias on piRNA pools by binding AGO3 in the Drosophila germline. Mol Cell 59: 553-563. PubMed ID: 26212455
Piwi-interacting RNAs (piRNAs) suppress transposon activity in animal germ cells. In the Drosophila ovary, primary Aubergine (Aub)-bound antisense piRNAs initiate the ping-pong cycle to produce secondary AGO3-bound sense piRNAs. This increases the number of secondary Aub-bound antisense piRNAs that can act to destroy transposon mRNAs. This study shows that Krimper (Krimp), a Tudor-domain protein, directly interacts with piRNA-free AGO3 to promote symmetrical dimethylarginine (sDMA) modification, ensuring sense piRNA-loading onto sDMA-modified AGO3. In aub mutant ovaries, AGO3 associates with ping-pong signature piRNAs, suggesting AGO3's compatibility with primary piRNA loading. Krimp sequesters ectopically expressed AGO3 within Krimp bodies in cultured ovarian somatic cells (OSCs), in which only the primary piRNA pathway operates. Upon krimp-RNAi in OSCs, AGO3 loads with piRNAs, further showing the capacity of AGO3 for primary piRNA loading. It is proposed that Krimp enforces an antisense bias on piRNA pools by binding AGO3 and blocking its access to primary piRNAs.

Webster, A., Li, S., Hur, J.K., Wachsmuth, M., Bois, J.S., Perkins, E.M., Patel, D.J. and Aravin, A.A. (2015). Aub and Ago3 are recruited to nuage through two mechanisms to form a ping-pong complex assembled by Krimper. Mol Cell 59: 564-575. PubMed ID: 26295961
In Drosophila, two Piwi proteins, Aubergine (Aub) and Argonaute-3 (Ago3), localize to perinuclear "nuage" granules and use guide piRNAs to target and destroy transposable element transcripts. This study finds that Aub and Ago3 are recruited to nuage by two different mechanisms. Aub requires a piRNA guide for nuage recruitment, indicating that its localization depends on recognition of RNA targets. Ago3 is recruited to nuage independently of a piRNA cargo and relies on interaction with Krimper, a stable component of nuage that is able to aggregate in the absence of other nuage proteins. It was shown that Krimper interacts directly with Aub and Ago3 to coordinate the assembly of the ping-pong piRNA processing (4P) complex. Symmetrical dimethylated arginines are required for Aub to interact with Krimper, but they are dispensable for Ago3 to bind Krimper. This study reveals a multi-step process responsible for the assembly and function of nuage complexes in piRNA-guided transposon repression.

Erwin, A. A., Galdos, M. A., Wickersheim, M. L., Harrison, C. C., Marr, K. D., Colicchio, J. M. and Blumenstiel, J. P. (2015). piRNAs are associated with diverse transgenerational effects on gene and transposon expression in a hybrid dysgenic syndrome of D. virilis. PLoS Genet 11: e1005332. PubMed ID: 26241928
Hybrid dysgenic syndromes are a strong form of genomic incompatibility that can arise when transposable element (TE) family abundance differs between two parents. When TEs inherited from the father are absent in the mother's genome, TEs can become activated in the progeny, causing germline damage and sterility. Studies in Drosophila indicate that dysgenesis can occur when TEs inherited paternally are not matched with a pool of corresponding TE silencing PIWI-interacting RNAs (piRNAs) provisioned by the female germline. Using the D. virilis syndrome of hybrid dysgenesis as a model, this study characterize the effects that divergence in TE profile between parents has on offspring. Overall, this study shows that divergence in the TE landscape is associated with persisting differences in germline TE expression when comparing genetically identical females of reciprocal crosses and these differences are transmitted to the next generation. Moreover, chronic and persisting TE expression coincides with increased levels of genic piRNAs associated with reduced gene expression. Gene expression is idiosyncratically influenced by differences in the genic piRNA profile of the parents that arise though polymorphic TE insertions. Overall, these results support a model in which early germline events in dysgenesis establish a chronic, stable state of both TE and gene expression in the germline that is maintained through adulthood and transmitted to the next generation.

Morgunova, V., Akulenko, N., Radion, E., Olovnikov, I., Abramov, Y., Olenina, L. V., Shpiz, S., Kopytova, D. V., Georgieva, S. G. and Kalmykova, A. (2015). Telomeric repeat silencing in germ cells is essential for early development in Drosophila. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 26240377
The germline-specific role of telomeres consists of chromosome end elongation and proper chromosome segregation during early developmental stages. Despite the crucial role of telomeres in germ cells, little is known about telomere biology in the germline. This study analyzed telomere homeostasis in the Drosophila female germline and early embryos. A novel germline-specific function of deadenylase complex Ccr4-Not in the telomeric transcript surveillance mechanism is reported. Depletion of Ccr4-Not complex components causes strong derepression of the telomeric retroelement HeT-A in the germ cells, accompanied by elongation of the HeT-A poly(A) tail. Dysfunction of transcription factors Woc and Trf2, as well as RNA-binding protein Ars2, also results in the accumulation of excessively polyadenylated HeT-A transcripts in ovaries. Germline knockdowns of Ccr4-Not components, Woc, Trf2 and Ars2, lead to abnormal mitosis in early embryos, characterized by chromosome missegregation, centrosome dysfunction and spindle multipolarity. Moreover, the observed phenotype is accompanied by the accumulation of HeT-A transcripts around the centrosomes in early embryos, suggesting the putative relationship between overexpression of telomeric transcripts and mitotic defects. These data demonstrate that Ccr4-Not, Woc, Trf2 and Ars2, components of different regulatory pathways, are required for telomere protection in the germline in order to guarantee normal development.

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