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

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

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

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

Saturday, March 14th

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

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

Couton, L., Mauss, A. S., Yunusov, T., Diegelmann, S., Evers, J. F. and Landgraf, M. (2015) Development of connectivity in a motoneuronal network in Drosophila larvae. Curr Biol 25: 568-576. PubMed ID: 25702582 Summary: Much of understanding of how neural networks develop is based on studies of sensory systems, revealing often highly stereotyped patterns of connections, particularly as these diverge from the presynaptic terminals of sensory neurons. Considerably less is known about the wiring strategies of motor networks, where connections converge onto the dendrites of motoneurons. This study investigated patterns of synaptic connections between identified motoneurons with sensory neurons and interneurons in the motor network of the Drosophila larva and how these change as it develops. As animals grow, motoneurons were found to increase the number of synapses with existing presynaptic partners. Different motoneurons form characteristic cell-type-specific patterns of connections. At the same time, there is considerable variability in the number of synapses formed on motoneuron dendrites, which contrasts with the stereotypy reported for presynaptic terminals of sensory neurons. Where two motoneurons of the same cell type contact a common interneuron partner, each postsynaptic cell can arrive at a different connectivity outcome. Experimentally changing the positioning of motoneuron dendrites shows that the geography of dendritic arbors in relation to presynaptic partner terminals is an important determinant in shaping patterns of connectivity. It is concluded that in the Drosophila larval motor network, the sets of connections that form between identified neurons manifest an unexpected level of variability. Synapse number and the likelihood of forming connections appear to be regulated on a cell-by-cell basis, determined primarily by the postsynaptic dendrites of motoneuron terminals.

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

Friday, March 13th

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

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

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

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

Thursday, March 12th

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

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

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

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

Wednesday, March 11th

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

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

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

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

Tuesday, March 10th

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

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

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

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

Monday, March 9th

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

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

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

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

Sunday, March 8th

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

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

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

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

Saturday, March 7th

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

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

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

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

Friday, March 6th

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

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

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

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

Thursday, March 5th

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

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

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

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

Wednesday, March 4th

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

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

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

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

Tuesday, March 3rd

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

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

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

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

Monday, March 2nd

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

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

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

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

Sunday, March 1st

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

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

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

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

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