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


Saturday, December 31st, 2016

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Mathur, V. and Schmidt, P.S. (2016). Adaptive patterns of phenotypic plasticity in laboratory and field environments in Drosophila melanogaster. Evolution [Epub ahead of print]. PubMed ID: 27925178
Identifying mechanisms of adaptation to variable environments is essential in developing a comprehensive understanding of evolutionary dynamics in natural populations. Phenotypic plasticity allows for phenotypic change in response to changes in the environment, and as such may play a major role in adaptation to environmental heterogeneity. This study examined the plasticity of stress response in D. melanogaster originating from two distinct geographic regions and ecological habitats. Adults were given a short-term, 5-day exposure to combinations of temperature and photoperiod to elicit a plastic response for three fundamental aspects of stress tolerance that vary adaptively with geography. This was replicated in both the laboratory and in outdoor enclosures in the field. In the laboratory, geographic origin was found to be the primary determinant of the stress response. Temperature and the interaction between temperature and photoperiod were also found to significantly affect stress resistance. In the outdoor enclosures, plasticity was distinct among traits and between geographic regions. These results demonstrate that short-term exposure of adults to ecologically relevant environmental cues results in predictable effects on multiple aspects of fitness. These patterns of plasticity vary among traits and are highly distinct between the two examined geographic regions, consistent with patterns of local adaptation to climate and associated environmental parameters. 

Robillard, E., Le Rouzic, A., Zhang, Z., Capy, P. and Hua-Van, A. (2016). Experimental evolution reveals hyperparasitic interactions among transposable elements. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27930288
Transposable elements (TEs) are repeated DNA sequences that can constitute a substantial part of genomes. Studying TEs' activity, interactions, and accumulation dynamics is thus of major interest to understand genome evolution. This study describes the transposition dynamics of cut-and-paste mariner elements during experimental (short- and longer-term) evolution in Drosophila melanogaster. Flies with autonomous and nonautonomous mariner copies were introduced in populations containing no active mariner, and TE accumulation was tracked by quantitative PCR for up to 100 generations. Ther results demonstrate that (i) active mariner elements are highly invasive and characterized by an elevated transposition rate, confirming their capacity to spread in populations, as predicted by the "selfish-DNA" mechanism; (ii) nonautonomous copies act as parasites of autonomous mariner elements by hijacking the transposition machinery produced by active mariner, which can be considered as a case of hyperparasitism; (iii) this behavior resulted in a failure of active copies to amplify which systematically drove the whole family to extinction in less than 100 generations. This study nicely illustrates how the presence of transposition-competitive variants can deeply impair TE dynamics and gives clues to the extraordinary diversity of TE evolutionary histories observed in genomes.
Kwan, C. W., Gavin-Smyth, J., Ferguson, E. L. and Schmidt-Ott, U. (2016). Functional evolution of a morphogenetic gradient. Elife 5 [Epub ahead of print]. PubMed ID: 28005004
Evolutionary Homolog Study
Bone Morphogenetic Proteins (BMPs) pattern the dorsal-ventral axis of bilaterian embryos; however, their roles in the evolution of body plan are largely unknown. This study examined their functional evolution in fly embryos. BMP signaling specifies two extraembryonic tissues, the serosa and amnion, in basal-branching flies such as Megaselia abdita, but only one, the amnioserosa, in Drosophila melanogaster. The BMP signaling dynamics are similar in both species until the beginning of gastrulation, when BMP signaling broadens and intensifies at the edge of the germ rudiment in Megaselia, while remaining static in Drosophila. This study shows that the differences in gradient dynamics and tissue specification result from evolutionary changes in the gene regulatory network that controls the activity of a positive feedback circuit on BMP signaling, involving the tumor necrosis factor alpha homolog eiger. These data illustrate an evolutionary mechanism by which spatiotemporal changes in morphogen gradients can guide tissue complexity.
Roux, C., Fraisse, C., Romiguier, J., Anciaux, Y., Galtier, N. and Bierne, N. (2016). Shedding light on the grey zone of speciation along a continuum of genomic divergence. PLoS Biol 14(12): e2000234. PubMed ID: 28027292
Speciation results from the progressive accumulation of mutations that decrease the probability of mating between parental populations or reduce the fitness of hybrids-the so-called species barriers. Of primary importance is the prevalence of gene flow between diverging entities, which is central in most species concepts and has been widely discussed in recent years. This study explored the continuum of speciation thanks to a comparative analysis of genomic data from 61 pairs of populations/species of animals with variable levels of divergence. The intermediate "grey zone" of speciation, in which taxonomy is often controversial, spans from 0.5% to 2% of net synonymous Divergence, irrespective of species life history traits or ecology. Thanks to appropriate modeling of among-locus variation in genetic drift and introgression rate, this study clarifies the status of the majority of ambiguous cases and uncovers a number of cryptic species. This analysis also reveals the high incidence in animals of semi-isolated species (when some but not all loci are affected by barriers to gene flow) and highlights the intrinsic difficulty, both statistical and conceptual, of delineating species in the grey zone of speciation.
Hollis, B., Keller, L. and Kawecki, T. J. (2016). Sexual selection shapes development and maturation rates in Drosophila. Evolution [Epub ahead of print]. PubMed ID: 27883363
Explanations for the evolution of delayed maturity usually invoke trade-offs mediated by growth, but processes of reproductive maturation continue long after growth has ceased. This study tested whether sexual selection shapes the rate of posteclosion maturation in the fruit fly Drosophila melanogaster. Populations maintained for more than 100 generations under a short generation time and polygamous mating system evolved faster posteclosion maturation and faster egg-to-adult development of males, when compared to populations kept under short generations and randomized monogamy that eliminated sexual selection. An independent assay demonstrated that more mature males have higher fitness under polygamy, but this advantage disappears under monogamy. In contrast, for females greater maturity was equally advantageous under polygamy and monogamy. Furthermore, monogamous populations evolved faster development and maturation of females relative to polygamous populations, with no detectable trade-offs with adult size or egg-to-adult survival. These results suggest that a major aspect of male maturation involves developing traits that increase success in sexual competition, whereas female maturation is not limited by investment in traits involved in mate choice or defense against male antagonism. Moreover, rates of juvenile development and adult maturation can readily evolve in opposite directions in the two sexes, possibly implicating polymorphisms with sexually antagonistic pleiotropy.
Gonzalez, P., Uhlinger, K. R. and Lowe, C. J. (2016). The adult body plan of indirect developing hemichordates develops by adding a Hox-patterned trunk to an anterior larval territory. Curr Biol [Epub ahead of print]. PubMed ID: 27939313
Evolutionary Homolog Study
Many animals are indirect developers with distinct larval and adult body plans. This study compared the larval and adult body plans of an indirect developing hemichordate, Schizocardium californicum. The expression is described of 27 transcription factors with conserved roles in deuterostome ectodermal anteroposterior (AP) patterning in developing embryos, tornaria larvae, and post-metamorphic juveniles and show that the tornaria larva of S. californicum is transcriptionally similar to a truncated version of the adult. The larval ectoderm has an anterior molecular signature, while most of the trunk, defined by the expression of hox1-7, is absent. Posterior ectodermal activation of Hox is initiated in the late larva prior to metamorphosis, in preparation for the transition to the adult form, in which the AP axis converges on a molecular architecture similar to that of the direct developing hemichordate Saccoglossus kowalevskii. These results identify a molecular correlate of a major difference in body plan between hemichordate larval and adult forms and confirm the hypothesis that deuterostome larvae are "swimming heads". This will allow future comparative studies with hemichordates to take into account molecular differences caused by early life history evolution within the phylum. Additionally, comparisons with other phyla suggest that a delay in trunk development is a feature of indirect development shared across distantly related phyla.

Friday, December 30th

Chen, X., Rahman, R., Guo, F. and Rosbash, M. (2016). Genome-wide identification of neuronal activity-regulated genes in Drosophila. Elife 5. PubMed ID: 27936378
Activity-regulated genes (ARGs) are important for neuronal functions like long-term memory and are well-characterized in mammals but poorly studied in other model organisms like Drosophila. This study stimulated fly neurons with different paradigms and identified ARGs using high-throughput sequencing from brains as well as from sorted neurons: they included a narrow set of circadian neurons as well as dopaminergic neurons. Surprisingly, many ARGs are specific to the stimulation paradigm and very specific to neuron type. In addition and unlike mammalian immediate early genes (IEGs), fly ARGs do not have short gene lengths and are less enriched for transcription factor function. Chromatin assays using ATAC-sequencing show that the transcription start sites (TSS) of ARGs do not change with neural firing but are already accessible prior to stimulation. Lastly based on binding site enrichment in ARGs, transcription factor mediators of firing were identified and neuronal activity reporters were created. Several of the induced transcription factors encode proteins with high sequence identity to well-known mammalian IEGs: HR38 shows 67% identity to nuclear receptor subfamily four group A member 2 (NR4A2); Stripe has 82% identity to early growth response protein 3 (EGR3); Cabut is 61% identical to Krueppel-like factor 11 (KLF11)
Perea, G., et al. (2016). Activity-dependent switch of GABAergic inhibition into glutamatergic excitation in astrocyte-neuron networks. Elife 5. PubMed ID: 28012274
Evolutionary Homolog Study
Interneurons are critical for proper neural network function and can activate Ca2+ signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, this study found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and the sign (inhibition vs potentiation) of the GABA-mediated effects were determined. While synaptic inhibition was mediated by GABAA receptors (see Drosophila Rdl), potentiation involved astrocyte GABAB receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABAB receptor (Gabbr1; see Drosophila metabotropic GABA-B receptor subtype 1) knockout mice, the glial source of the interneuron-induced potentiation was confirmed, and the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo was demonstrated. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay.
Yao, Z., Bennett, A.J., Clem, J.L. and Shafer, O.T. (2016). The Drosophila clock neuron network features diverse coupling modes and requires network-wide coherence for robust circadian rhythms. Cell Rep 17: 2873-2881. PubMed ID: 27974202
In animals, networks of clock neurons containing molecular clocks orchestrate daily rhythms in physiology and behavior. However, how various types of clock neurons communicate and coordinate with one another to produce coherent circadian rhythms is not well understood. This study investigated clock neuron coupling in the brain of Drosophila and demonstrates that the fly's various groups of clock neurons display unique and complex coupling relationships to core pacemaker neurons. Furthermore, coordinated free-running rhythms require molecular clock synchrony not only within the well-characterized lateral clock neuron classes but also between lateral clock neurons and dorsal clock neurons. These results uncover unexpected patterns of coupling in the clock neuron network and reveal that robust free-running behavioral rhythms require a coherence of molecular oscillations across most of the fly's clock neuron network.

Li, L., Sanchez, C. P., Slaughter, B. D., Zhao, Y., Khan, M. R., Unruh, J. R., Rubinstein, B. and Si, K. (2016). A putative biochemical engram of long-term memory. Curr Biol 26(23): 3143-3156. PubMed ID: 27818176
How a transient experience creates an enduring yet dynamic memory remains an unresolved issue in studies of memory. Experience-dependent aggregation of the RNA-binding protein CPEB/Orb2 is one of the candidate mechanisms of memory maintenance. Using tools that allow rapid and reversible inactivation of Orb2 protein in neurons, this study found that Orb2 activity is required for encoding and recall of memory. From a screen, a DNA-J family chaperone, JJJ2, was identified that facilitates Orb2 aggregation, and ectopic expression of JJJ2 enhances the animal's capacity to form long-term memory. Tools were developed to visualize training-dependent aggregation of Orb2. Aggregated Orb2 in a subset of mushroom body neurons can serve as a "molecular signature" of memory and predict memory strength. These data indicate that self-sustaining aggregates of Orb2 may serve as a physical substrate of memory and provide a molecular basis for the perduring yet malleable nature of memory.

Yamagata, N., Hiroi, M., Kondo, S., Abe, A. and Tanimoto, H. (2016). Suppression of dopamine neurons mediates reward. PLoS Biol 14: e1002586. PubMed ID: 27997541
Massive activation of dopamine neurons is critical for natural reward and drug abuse. In contrast, the significance of their spontaneous activity remains elusive. In Drosophila melanogaster, depolarization of the protocerebral anterior medial (PAM) cluster dopamine neurons en masse signals reward to the mushroom body (MB) and drives appetitive memory. Focusing on the functional heterogeneity of PAM cluster neurons, this study identified that a single class of PAM neurons, PAM-γ3, mediates sugar reward by suppressing their own activity. PAM-γ3 is selectively required for appetitive olfactory learning, while activation of these neurons in turn induces aversive memory. Ongoing activity of PAM-γ3 gets suppressed upon sugar ingestion. Strikingly, transient inactivation of basal PAM-γ3 activity can substitute for reward and induces appetitive memory. Furthermore, the satiety-signaling neuropeptide Allatostatin A (AstA) is a key mediator that conveys inhibitory input onto PAM-γ3. These results suggest the significance of basal dopamine release in reward signaling and reveal a circuit mechanism for negative regulation

Mena, W., Diegelmann, S., Wegener, C. and Ewer, J. (2016). Stereotyped responses of Drosophila peptidergic neuronal ensemble depend on downstream neuromodulators. Elife 5. PubMed ID: 27976997
Neuropeptides play a key role in the regulation of behaviors and physiological responses including alertness, social recognition, and hunger, yet, their mechanism of action is poorly understood. This study focused on the endocrine control ecdysis behavior, which is used by arthropods to shed their cuticle at the end of every molt. Ecdysis is triggered by ETH (Ecdysis triggering hormone), and the response of peptidergic neurons that produce CCAP (crustacean cardioactive peptide), which are key targets of ETH and control the onset of ecdysis behavior, was shown to depend fundamentally on the actions of neuropeptides produced by other direct targets of ETH and released in a broad paracrine manner within the CNS; by autocrine influences from the CCAP neurons themselves; and by inhibitory actions mediated by GABA. These findings provide insights into how this critical insect behavior is controlled and general principles for understanding how neuropeptides organize neuronal activity and behaviors.

Thursday, December 29th

Nitta, Y., Yamazaki, D., Sugie, A., Hiroi, M. and Tabata, T. (2016). DISCO Interacting Protein 2 regulates axonal bifurcation and guidance of Drosophila mushroom body neurons. Dev Biol [Epub ahead of print]. PubMed ID: 27908785
Axonal branching is one of the key processes within the enormous complexity of the nervous system to enable a single neuron to send information to multiple targets. However, the molecular mechanisms that control branch formation are poorly understood. In particular, previous studies have rarely addressed the mechanisms underlying axonal bifurcation, in which axons form new branches via splitting of the growth cone. This study demonstrates that DISCO Interacting Protein 2 (DIP2) is required for precise axonal bifurcation in Drosophila mushroom body (MB) neurons by suppressing ectopic bifurcation and regulating the guidance of sister axons. DIP2 localizes to the plasma membrane. Domain function analysis revealed that the AMP-synthetase domains of DIP2 are essential for its function, which may involve exerting a catalytic activity that modifies fatty acids. Genetic analysis and subsequent biochemical analysis suggested that DIP2 is involved in the fatty acid metabolization of acyl-CoA. Taken together, these results reveal a function of DIP2 in the developing nervous system and provide a potential functional relationship between fatty acid metabolism and axon morphogenesis.
Tong, H., Li, Q., Zhang, Z. C., Li, Y. and Han, J. (2016). Neurexin regulates nighttime sleep by modulating synaptic transmission. Sci Rep 6: 38246. PubMed ID: 27905548
Neurexins are cell adhesion molecules involved in synaptic formation and synaptic transmission. Mutations in neurexin genes are linked to autism spectrum disorders (ASDs), which are frequently associated with sleep problems. However, the role of neurexin-mediated synaptic transmission in sleep regulation is unclear. This study shows that lack of the Drosophila α-neurexin homolog (FlyBase: Nrx-1) significantly reduces the quantity and quality of nighttime sleep and impairs sleep homeostasis. Neurexin expression in Drosophila mushroom body (MB) αβ neurons is essential for nighttime sleep. Reduced nighttime sleep in neurexin mutants is due to impaired αβ neuronal output, and neurexin functionally couples calcium channels (Cac) to regulate synaptic transmission. Finally, it was determined that αβ surface (αβs) neurons release both acetylcholine and short neuropeptide F (sNPF), whereas αβ core (αβc) neurons release sNPF to promote nighttime sleep. These findings reveal that neurexin regulates nighttime sleep by mediating the synaptic transmission of αβ neurons. This study elucidates the role of synaptic transmission in sleep regulation, and might offer insights into the mechanism of sleep disturbances in patients with autism disorders.
Watanabe, K., Furumizo, Y., Usui, T., Hattori, Y. and Uemura, T. (2016). Nutrient-dependent increased dendritic arborization of somatosensory neurons. Genes Cells [Epub ahead of print]. PubMed ID: 27868313
Suboptimal nutrition imposes developmental constraints on infant animals, which marshal adaptive responses to eventually become mature adults. Such responses are mounted at multiple levels from systemic to cellular. Little is known about how growth of postmitotic and morphologically complex cells, such as neurons, is controlled by nutritional status. This question was addressed using Class I and Class IV dendritic arborization neurons in Drosophila larvae. Class IV neurons have been shown to sense nociceptive thermal, mechanical and light stimuli, whereas Class I neurons are proprioceptors. Larvae were reared on diets with different protein and carbohydrate content throughout larval stages, and how morphologies of Class I or Class IV neurons were affected was examined. Dendritic arbors of Class IV neurons became more complex when larvae were reared on a low-yeast diet, which contains lower amounts of amino acids and other ingredients, compared to a high-yeast diet. In contrast, such low-yeast-dependent hyperarborization was not seen in Class I neurons. The physiological and metabolic implications of the hyperarborization phenotype are discussed in relation to a recent hypothesis that Class IV neurons sense protein-deficient stress and to this characterization of how the dietary yeast contents impacted larval metabolism.
Xu, L., He, J., Kaiser, A., Graber, N., Schlager, L., Ritze, Y. and Scholz, H. (2016). A single pair of serotonergic neurons counteracts serotonergic inhibition of ethanol attraction in Drosophila. PLoS One 11(12): e0167518. PubMed ID: 27936023
Attraction to ethanol is common in both flies and humans, but the neuromodulatory mechanisms underlying this innate attraction are not well understood. This study dissected the function of the key regulator of serotonin signaling - the serotonin transporter - in innate olfactory attraction to ethanol in Drosophila melanogaster. A mutated version of the serotonin transporter was generated that prolongs serotonin signaling in the synaptic cleft and is targeted via the Gal4 system to different sets of serotonergic neurons. Four serotonergic neurons were identified that inhibit the olfactory attraction to ethanol, and two additional neurons were identified that counteract this inhibition by strengthening olfactory information. These results reveal that compensation can occur on the circuit level and that serotonin has a bidirectional function in modulating the innate attraction to ethanol. Given the evolutionarily conserved nature of the serotonin transporter and serotonin, the bidirectional serotonergic mechanisms delineate a basic principle for how random behavior is switched into targeted approach behavior.

Peng, F. and Chittka, L. (2016). A simple computational model of the bee mushroom body can explain seemingly complex forms of olfactory learning and memory. Curr Biol [Epub ahead of print]. PubMed ID: 28017607
Evolutionary Homolog Study
Honeybees are models for studying how animals with relatively small brains accomplish complex cognition, displaying seemingly advanced (or "non-elemental") learning phenomena involving multiple conditioned stimuli. These include "peak shift"-where animals not only respond to entrained stimuli, but respond even more strongly to similar ones that are farther away from non-rewarding stimuli. Bees also display negative and positive patterning discrimination, responding in opposite ways to mixtures of two odors than to individual odors. Since Pavlov, it has often been assumed that such phenomena are more complex than simple associate learning. This paper presents a model of connections between olfactory sensory input and bees' mushroom bodies, incorporating empirically determined properties of mushroom body circuitry (random connectivity, sparse coding, and synaptic plasticity). The model's parameters were not optimized to replicate specific behavioral phenomena, because the study was interested in the emergent cognitive capacities that would pop out of a network constructed solely based on empirical neuroscientific information and plausible assumptions for unknown parameters. The circuitry mediating "simple" associative learning was shown to also replicate the various non-elemental forms of learning mentioned above and can effectively multi-task by replicating a range of different learning feats. It was found that projection neuron-kenyon cell (PN-KC) synaptic plasticity is crucial in controlling the generalization-discrimination trade-off - it facilitates peak shift and hinders patterning discrimination - and that PN-to-KC connection number can affect this trade-off. These findings question the notion that forms of learning that have been regarded as "higher order" are computationally more complex than "simple" associative learning.
Bedont, J. L., LeGates, T. A., Buhr, E., Bathini, A., Ling, J. P., Bell, B., Wu, M. N., Wong, P. C., Van Gelder, R. N., Mongrain, V., Hattar, S. and Blackshaw, S. (2016). An LHX1-regulated transcriptional network controls sleep/wake coupling and thermal resistance of the central circadian clockworks. Curr Biol [Epub ahead of print]. PubMed ID: 28017605
Evolutionary Homolog Study
The suprachiasmatic nucleus (SCN) is the central circadian clock in mammals. It is entrained by light but resistant to temperature shifts that entrain peripheral clocks. The SCN expresses many functionally important neuropeptides, including vasoactive intestinal peptide (VIP), which drives light entrainment, synchrony, and amplitude of SCN cellular clocks and organizes circadian behavior. The transcription factor LHX1(see Drosophila Lim1) drives SCN Vip expression, and cellular desynchrony in Lhx1-deficient SCN largely results from Vip loss. LHX1 regulates many genes other than Vip, yet activity rhythms in Lhx1-deficient mice are similar to Vip-/- mice under light-dark cycles and only somewhat worse in constant conditions. It is thought that LHX1 targets other than Vip have circadian functions overlooked in previous studies. This study compared circadian sleep and temperature rhythms of Lhx1- and Vip-deficient mice and found loss of acute light control of sleep in Lhx1 but not Vip mutants. Loss of circadian resistance to fever was also found in Lhx1 but not Vip mice that was partially recapitulated by heat application to cultured Lhx1-deficient SCN. Having identified VIP-independent functions of LHX1, the VIP-independent transcriptional network downstream of LHX1 and a largely separable VIP-dependent transcriptional network were also mapped. The VIP-independent network does not affect core clock amplitude and synchrony, unlike the VIP-dependent network. These studies identify Lhx1 as the first gene required for temperature resistance of the SCN clockworks and demonstrate that acute light control of sleep is routed through the SCN and its immediate output regions.

Wednesday, November 28th

Zhou, J., Yu, W. and Hardin, P. E. (2016). CLOCKWORK ORANGE enhances PERIOD mediated rhythms in transcriptional repression by antagonizing E-box binding by CLOCK-CYCLE. PLoS Genet 12: e1006430. PubMed ID: 27814361
The Drosophila circadian oscillator controls daily rhythms in physiology, metabolism and behavior via transcriptional feedback loops. CLOCK-CYCLE (CLK-CYC) heterodimers initiate feedback loop function by binding E-box elements to activate per and tim transcription. PER-TIM heterodimers then accumulate, bind CLK-CYC to inhibit transcription, and are ultimately degraded to enable the next round of transcription. The timing of transcriptional events in this feedback loop coincide with, and are controlled by, rhythms in CLK-CYC binding to E-boxes. PER rhythmically binds CLK-CYC to initiate transcriptional repression, and subsequently promotes the removal of CLK-CYC from E-boxes. However, little is known about the mechanism by which CLK-CYC is removed from DNA. Previous studies demonstrated that the transcription repressor CLOCKWORK ORANGE (CWO) contributes to core feedback loop function by repressing per and tim transcription in cultured S2 cells and in flies. This study shows that CWO rhythmically binds E-boxes upstream of core clock genes in a reciprocal manner to CLK, thereby promoting PER-dependent removal of CLK-CYC from E-boxes, and maintaining repression until PER is degraded and CLK-CYC displaces CWO from E-boxes to initiate transcription. These results suggest a model in which CWO co-represses CLK-CYC transcriptional activity in conjunction with PER by competing for E-box binding once CLK-CYC-PER complexes have formed. Given that CWO orthologs DEC1 and DEC2 also target E-boxes bound by CLOCK-BMAL1, a similar mechanism may operate in the mammalian clock.
Percival-Smith, A. (2016). Non-specificity of transcription factor function in Drosophila melanogaster. Dev Genes Evol [Epub ahead of print]. PubMed ID: 27848019
A major problem in developmental genetics is how HOX transcription factors, like Proboscipedia (PB) and Ultrabithorax (UBX), regulate distinct programs of gene expression to result in a proboscis versus a haltere, respectively, when the DNA-binding homeodomain (HD) of HOX transcription factors recognizes similar DNA-binding sequences. Indeed, the lack of DNA-binding specificity is a problem for all transcription factors (TFs), as the DNA-binding domains generally recognize small targets of five to six bases in length. Although not the initial intent of the study, extensive non-specificity of TF function were found. Multiple TFs including HOX and HD-containing and non-HD-containing TFs induced both wingless and eyeless phenotypes. The TFs Labial (LAB), Deformed (DFD), Fushi tarazu (FTZ), and Squeeze (SQZ) induced ectopic larval thoracic (T) 1 beard formation in T2 and T3. The TF Doublesex male (DSXM) rescued the reduced maxillary palp pb phenotype. These examples of non-specificity of TF function across classes of TFs, combined with previous observations, compromise the implicit, initial assumption often made that an intrinsic mechanism of TF specificity is important for function. Interestingly, the functional complementation of the pb phenotype may suggest a larger role for regulation of expression of TFs in restriction of function as opposed to an intrinsic specificity of TF function. These observations have major ramifications for analysis of functional conservation in evolution and development.
Seyres, D., Ghavi-Helm, Y., Junion, G., Taghli-Lamallem, O., Guichard, C., Roder, L., Girardot, C., Furlong, E. E. and Perrin, L. (2016). Identification and in silico modeling of enhancers reveals new features of the cardiac differentiation network. Development 143(23): 4533-4542. PubMed ID: 27899510
Developmental patterning and tissue formation are regulated through complex gene regulatory networks (GRNs) driven through the action of transcription factors (TFs) converging on enhancer elements. As a point of entry to dissect the poorly defined GRN underlying cardiomyocyte differentiation, an integrated approach was appled to identify active enhancers and TFs involved in Drosophila heart development. The Drosophila heart consists of 104 cardiomyocytes, representing less than 0.5% of all cells in the embryo. By modifying BiTS-ChIP for rare cells, H3K4me3 and H3K27ac chromatin landscapes were examined to identify active promoters and enhancers specifically in cardiomyocytes. These in vivo data were complemented by a machine learning approach and extensive in vivo validation in transgenic embryos, which identified many new heart enhancers and their associated TF motifs. These results implicate many new TFs in late stages of heart development, including Bagpipe, an Nkx3.2 ortholog, which is shown to be essential for differentiated heart function.
Symmons, O., Pan, L., Remeseiro, S., Aktas, T., Klein, F., Huber, W. and Spitz, F. (2016). The Shh topological domain facilitates the action of remote enhancers by reducing the effects of genomic distances. Dev Cell 39: 529-543. PubMed ID: 27867070
Evolutionary Homolog Study:
Gene expression often requires interaction between promoters and distant enhancers, which occur within the context of highly organized topologically associating domains (TADs) (see Drosophila chromatin organization). Using a series of engineered chromosomal rearrangements at the Shh (see Drosophila hh) locus, this study carried out an extensive fine-scale characterization of the factors that govern the long-range regulatory interactions controlling Shh expression. It was shown that Shh enhancers act pervasively, yet not uniformly, throughout the TAD. Importantly, changing intra-TAD distances have no impact on Shh expression. In contrast, inversions disrupting the TAD alter global folding of the region and prevent regulatory contacts in a distance-dependent manner. Data indicate that the Shh TAD promotes distance-independent contacts between distant regions that would otherwise interact only sporadically, enabling functional communication between them. In large genomes where genomic distances per se can limit regulatory interactions, this function of TADs could be as essential for gene expression as the formation of insulated neighborhoods.

Tuesday, December 27th

Silva, D., Olsen, K. W., Bednarz, M. N., Droste, A., Lenkeit, C. P., Chaharbakhshi, E., Temple-Wood, E. R. and Jemc, J. C. (2016). Regulation of gonad morphogenesis in Drosophila melanogaster by BTB family transcription factors. PLoS One 11(11): e0167283. PubMed ID: 27898696
During embryogenesis, primordial germ cells (PGCs) and somatic gonadal precursor cells (SGPs) migrate and coalesce to form the early gonad. A failure of the PGCs and SGPs to form a gonad with the proper architecture not only affects germ cell development, but can also lead to infertility. Therefore, it is critical to identify the molecular mechanisms that function within both the PGCs and SGPs to promote gonad morphogenesis. This study has characterized the phenotypes of two genes, longitudinals lacking (lola) and ribbon (rib), that are required for the coalescence and compaction of the embryonic gonad in Drosophila melanogaster. rib and lola are expressed in the SGPs of the developing gonad, and genetic interaction analysis suggests these proteins cooperate to regulate gonad development. Both genes encode proteins with DNA binding motifs and a conserved protein-protein interaction domain, known as the Broad complex, Tramtrack, Bric-a-brac (BTB) domain. Through molecular modeling and yeast-two hybrid studies, it was demonstrated that Rib and Lola homo- and heterodimerize via their BTB domains. In addition, analysis of the colocalization of Rib and Lola with marks of transcriptional activation and repression on polytene chromosomes reveals that Rib and Lola colocalize with both repressive and activating marks and with each other. While previous studies have identified Rib and Lola targets in other tissues, Rib and Lola are likely to function via different downstream targets in the gonad. These results suggest that Rib and Lola act as dual-function transcription factors to cooperatively regulate embryonic gonad morphogenesis.
Dranow, D.B., Hu, K., Bird, A.M., Lawry, S.T., Adams, M.T., Sanchez, A., Amatruda, J.F. and Draper, B.W. (2016). Bmp15 is an oocyte-produced signal required for maintenance of the adult female sexual phenotype in zebrafish. PLoS Genet 12: e1006323. PubMed ID: 27642754
Evolutionary Homolog Study:
Although the zebrafish is a major model organism, how they determine sex (see Drosophila sex determination) is not well understood. In domesticated zebrafish, sex determination appears to be polygenic, being influenced by multiple genetic factors that may vary from strain to strain, and additionally can be influenced by environmental factors. However, the requirement of germ cells for female sex determination is well documented: animals that lack germ cells, or oocytes in particular, develop exclusively as males. Recently, it has been determined that oocytes are also required throughout the adult life of the animal to maintain the differentiated female state. How oocytes control sex differentiation and maintenance of the sexual phenotype is unknown. This study generated targeted mutations in genes for two oocyte produced signaling molecules, Bmp15 and Gdf9 (see Drosophila Dpp) and found a novel role for Bmp15 in maintaining adult female sex differentiation in zebrafish. Females deficient in Bmp15 begin development normally but switch sex during the mid- to late- juvenile stage, and become fertile males. Additionally, by generating mutations in the aromatase cyp19a1a (see Drosophila Cyp311a1), it was shown that estrogen production is necessary for female development and that the function of Bmp15 in female sex maintenance is likely linked to the regulation of estrogen biosynthesis via promoting the development of estrogen-producing granulosa cells in the oocyte follicle.
Hurd, T.R., Herrmann, B., Sauerwald, J., Sanny, J., Grosch, M. and Lehmann, R. (2016). Long Oskar controls mitochondrial inheritance in Drosophila melanogaster. Dev Cell 39: 560-571. PubMed ID: 27923120
Inherited mtDNA mutations cause severe human disease. In most species, mitochondria are inherited maternally through mechanisms that are poorly understood. Genes that specifically control the inheritance of mitochondria in the germline are unknown. This study shows that the long isoform of the protein Oskar regulates the maternal inheritance of mitochondria in Drosophila melanogaster. During oogenesis mitochondria accumulate at the oocyte posterior, concurrent with the bulk streaming and churning of the oocyte cytoplasm. Long Oskar traps and maintains mitochondria at the posterior at the site of primordial germ cell (PGC) formation through an actin-dependent mechanism. Mutating long oskar strongly reduces the number of mtDNA molecules inherited by PGCs. Therefore, Long Oskar ensures germline transmission of mitochondria to the next generation. These results provide molecular insight into how mitochondria are passed from mother to offspring, as well as how they are positioned and asymmetrically partitioned within polarized cells.

Cattenoz, P. B., Delaporte, C., Bazzi, W. and Giangrande, A. (2016). An evolutionary conserved interaction between the Gcm transcription factor and the SF1 nuclear receptor in the female reproductive system. Sci Rep 6: 37792. PubMed ID: 27886257
NR5A1 is essential for the development and for the function of steroid producing glands of the reproductive system. Moreover, its misregulation is associated with endometriosis, which is the first cause of infertility in women. Hr39, the Drosophila ortholog of NR5A1, is expressed and required in the secretory cells of the spermatheca, the female exocrine gland that ensures fertility by secreting substances that attract and capacitate the spermatozoids.This study has identify a direct regulator of Hr39 in the spermatheca: the Gcm transcription factor. Furthermore, lack of Gcm prevents the production of the secretory cells and leads to female sterility in Drosophila. Hr39 regulation by Gcm seems conserved in mammals and involves the modification of the DNA methylation profile of mNr5a1. This study identifies a new molecular pathway in female reproductive system development and suggests a role for hGCM in the progression of reproductive tract diseases in humans.

Monday, December 26

Wang, Z., Tacchelly-Benites, O., Yang, E. and Ahmed, Y. (2016). Dual roles for membrane association of Drosophila Axin in Wnt signaling. PLoS Genet 12: e1006494. PubMed ID: 27959917
Axin, a concentration-limiting scaffold protein, facilitates assembly of a "destruction complex" that prevents Wnt signaling in the unstimulated state and a plasma membrane-associated "signalosome" that activates signaling following Wnt stimulation. In the classical model, Axin is cytoplasmic under basal conditions, but relocates to the cell membrane after Wnt exposure. This study analyzed the subcellular distribution of endogenous Drosophila Axin in vivo and found that a pool of Axin localizes to cell membrane proximal puncta even in the absence of Wnt stimulation. Axin localization in these puncta is dependent on the destruction complex component Adenomatous polyposis coli (Apc). In the unstimulated state, the membrane association of Axin increases its Tankyrase-dependent ADP-ribosylation and consequent proteasomal degradation to control its basal levels. Furthermore, Wnt stimulation does not result in a bulk redistribution of Axin from cytoplasmic to membrane pools, but causes an initial increase of Axin in both of these pools, with concomitant changes in two post-translational modifications, followed by Axin proteolysis hours later. Finally, the ADP-ribosylated Axin that increases rapidly following Wnt stimulation is membrane associated. The study concludes that even in the unstimulated state, a pool of Axin forms membrane-proximal puncta that are dependent on Apc, and that membrane association regulates both Axin levels and Axin's role in the rapid activation of signaling that follows Wnt exposure.

Lim, B., Dsilva, C. J., Kevrekidis, I. G. and Shvartsman, S. Y. (2017). Reconstructing ERK signaling in the Drosophila embryo from fixed images. Methods Mol Biol 1487: 337-351. PubMed ID: 27924579
The early Drosophila embryo provides unique opportunities for quantitative studies of ERK signaling. This system is characterized by simple anatomy, the ease of obtaining large numbers of staged embryos, and the availability of powerful tools for genetic manipulation of the ERK pathway. This paper describes how these experimental advantages can be combined with recently developed microfluidic devices for high throughput imaging of ERK activation dynamics. Focus was placed on the stage during the third hour of development, when ERK activation is essential for patterning of the future nerve cord. This approach starts with an ensemble of fixed embryos stained with an antibody that recognizes the active, dually phosphorylated form of ERK. Each embryo in this ensemble provides a snapshot of the spatial and temporal pattern of ERK activation during development. Then the ages of fixed embryos are quantitatively estimated using a model that links their morphology and developmental time. This model is learned based on live imaging of cellularization and gastrulation, two highly stereotyped morphogenetic processes at this stage of embryogenesis. Applying this approach, ERK signaling can be characterized at high spatial and temporal resolution. This methodology can be readily extended to studies of ERK regulation and function in multiple mutant backgrounds, providing a versatile assay for quantitative studies of developmental ERK signaling.
Abe, Y., Sako, K., Takagaki, K., Hirayama, Y., Uchida, K.S., Herman, J.A., DeLuca, J.G. and Hirota, T. (2016). HP1-assisted Aurora B kinase activity prevents chromosome segregation errors. Dev Cell 36: 487-497. PubMed ID: 26954544
Evolutionary Homolog Study:
Incorrect attachment of kinetochore microtubules is the leading cause of chromosome missegregation in cancers. The highly conserved chromosomal passenger complex (CPC) (see Drosophila cell cycle), containing mitotic kinase Aurora B (see Drosophila aurora B) as a catalytic subunit, ensures faithful chromosome segregation through destabilizing incorrect microtubule attachments and promoting biorientation of chromosomes on the mitotic spindle. It is unknown whether CPC dysfunction affects chromosome segregation fidelity in cancers and, if so, how. This study shows that heterochromatin protein 1 (HP1) (see Drosophila Suppressor of variegation 205) is an essential CPC component required for full Aurora B activity. HP1 binding to the CPC becomes particularly important when Aurora B phosphorylates kinetochore targets to eliminate erroneous microtubule attachments. Remarkably, a reduced proportion of HP1 bound to CPC is widespread in cancers, which causes an impairment in Aurora B activity. These results indicate that HP1 is an essential modulator for CPC function and identify a molecular basis for chromosome segregation errors in cancer cells.

Liu, S., Sun, J., Wang, D., Pflugfelder, G. O. and Shen, J. (2016). Fold formation at the compartment boundary of Drosophila wing requires Yki signaling to suppress JNK dependent apoptosis. Sci Rep 6: 38003. PubMed ID: 27897227
Compartment boundaries prevent cell populations of different lineage from intermingling. In many cases, compartment boundaries are associated with morphological folds. However, in the Drosophila wing imaginal disc, fold formation at the anterior/posterior (A/P) compartment boundary is suppressed, probably as a prerequisite for the formation of a flat wing surface. Fold suppression depends on optomotor-blind (omb). Omb mutant animals develop a deep apical fold at the A/P boundary of the larval wing disc and an A/P cleft in the adult wing. A/P fold formation is controlled by different signaling pathways. Jun N-terminal kinase (JNK) and Yorkie (Yki) signaling are activated in cells along the fold and are necessary for the A/P fold to develop. While JNK promotes cell shape changes and cell death, Yki target genes are required to antagonize apoptosis, explaining why both pathways need to be active for the formation of a stable fold.
Tarchini, B., Tadenev, A. L., Devanney, N. and Cayouette, M. (2016). A link between planar polarity and staircase-like bundle architecture in hair cells. Development 143(21): 3926-3932. PubMed ID: 27660326
Evolutionary Homolog Study
Sensory perception in the inner ear relies on the hair bundle, the highly polarized brush of movement detectors that crowns hair cells. Previous studies have shown that, in the mouse cochlea, the edge of the forming bundle is defined by the 'bare zone', a microvilli-free sub-region of apical membrane specified by the Insc-LGN-Gαi protein complex. This study reports that LGN (see Drosophila Partner of inscuteable) and Gαi (see Drosophila G protein αi subunit 65A) also occupy the very tip of stereocilia that directly abut the bare zone. LGN and Gαi are both essential for promoting the elongation and differential identity of stereocilia across rows. Interestingly, it was also revealed that total LGN-Gαi protein amounts are actively balanced between the bare zone and stereocilia tips, suggesting that early planar asymmetry of protein enrichment at the bare zone confers adjacent stereocilia their tallest identity. It is proposed that LGN and Gαi participate in a long-inferred signal that originates outside the bundle to model its staircase-like architecture, a property that is essential for direction sensitivity to mechanical deflection and hearing.
Green, Y. S., Kwon, S., Mimoto, M. S., Xie, Y. and Christian, J. L. (2016). Tril targets Smad7 for degradation to allow hematopoietic specification in Xenopus embryos. Development 143(21): 4016-4026. PubMed ID: 27633996
Evolutionary Homolog Study
In Xenopus laevis, bone morphogenetic proteins (Bmps) induce expression of the transcription factor Gata2 (see Drosophila Serpent) during gastrulation, and Gata2 is required in both ectodermal and mesodermal cells to enable mesoderm to commit to a hematopoietic fate. This study identified tril as a Gata2 target gene that is required in both ectoderm and mesoderm for primitive hematopoiesis to occur. Tril is a transmembrane protein that functions as a co-receptor for Toll-like receptors to mediate innate immune responses in the adult brain, but developmental roles for this molecule have not been identified. This study shows that Tril function is required both upstream and downstream of Bmp receptor-mediated Smad1 (see Drosophila Mad) phosphorylation for induction of Bmp target genes. Mechanistically, Tril triggers degradation of the Bmp inhibitor Smad7. Tril-dependent downregulation of Smad7 relieves repression of endogenous Bmp signaling during gastrulation and this enables mesodermal progenitors to commit to a blood fate. Thus, Tril is a novel component of a Bmp-Gata2 positive-feedback loop that plays an essential role in hematopoietic specification.

Sunday, December 25th

Hermle, T., Braun, D.A., Helmstädter, M., Huber, T.B. and Hildebrandt, F. (2016). Modeling monogenic human nephrotic syndrome in the Drosophila garland cell nephrocyte. J Am Soc Nephrol [Epub ahead of print]. PubMed ID: 27932481
Steroid-resistant nephrotic syndrome is characterized by podocyte dysfunction. Drosophila garland cell nephrocytes are podocyte-like cells and thus provide a potential in vivo model in which to study the pathogenesis of nephrotic syndrome. However, relevant pathomechanisms of nephrotic syndrome have not been studied in nephrocytes. This study discovered that two Drosophila slit diaphragm proteins, orthologs of the human genes encoding nephrin and nephrin-like protein 1, colocalize within a fingerprint-like staining pattern that correlates with ultrastructural morphology. Using RNAi and conditional CRISPR/Cas9 in nephrocytes, it was found that this pattern depends on the expression of both orthologs. Tracer endocytosis by nephrocytes requires Cubilin and reflects size selectivity analogous to that of glomerular function. Using RNAi and tracer endocytosis as a functional read-out, Drosophila orthologs of human monogenic causes of nephrotic syndrome were screened and conservation of the central pathogenetic alterations was observed. It was found that the silencing of the coenzyme Q10 (CoQ10) biosynthesis gene Coq2 disrupts slit diaphragm morphology. Restoration of CoQ10 synthesis by vanillic acid partially rescues the phenotypic and functional alterations induced by Coq2-RNAi. Notably, Coq2 colocalizes with mitochondria, and Coq2 silencing increases the formation of reactive oxygen species (ROS). Silencing of ND75, a subunit of the mitochondrial respiratory chain that controls ROS formation independently of CoQ10, phenocopies the effect of Coq2-RNAi. Moreover, the ROS scavenger glutathione partially rescues the effects of Coq2-RNAi. In conclusion, Drosophila garland cell nephrocytes provide a model with which to study the pathogenesis of nephrotic syndrome, and ROS formation may be a pathomechanism of COQ2-nephropathy.

M'Angale, P. G. and Staveley, B. E. (2016). Loss of porin function in dopaminergic neurons of Drosophila is suppressed by Buffy. J Biomed Sci 23(1): 84. PubMed ID: 27881168
Mitochondrial porin, also known as the voltage-dependent anion channel (VDAC), is a multi-functional channel protein that shuttles metabolites between the mitochondria and the cytosol and implicated in cellular life and death decisions. The inhibition of porin under the control of neuronal Ddc-Gal4 result in short lifespan and in an age-dependent loss in locomotor function, phenotypes that are strongly associated with Drosophila models of Parkinson disease. Loss of porin function was achieved through exploitation of RNAi while derivative lines were generated by homologous recombination and tested by PCR. The expression of human α-synuclein transgene in neuronal populations that include dopamine producing neurons under the control of Ddc-Gal4 produces a robust Parkinson disease model, and results in severely reduced lifespan and locomotor dysfunction. In addition, the porin-induced phenotypes are greatly suppressed when the pro-survival Bcl-2 homologue Buffy is overexpressed in these neurons and in the developing eye adding to the cellular advantages of altered expression of this anti-apoptotic gene. When α-synuclein was co-expressed along with porin, it results in a decrease in lifespan and impaired climbing ability. This enhancement of the α-synuclein-induced phenotypes observed in neurons was demonstrated in the neuron rich eye, where the simultaneous co-expression of porin-RNAi and α-synuclein resulted in an enhanced eye phenotype, marked by reduced number of ommatidia and increased disarray of the ommatidia. It is concluded that the inhibition of porin in dopaminergic neurons among others results in reduced lifespan and age-dependent loss in climbing ability, phenotypes that are suppressed by the overexpression of the sole pro-survival Bcl-2 homologue Buffy. The inhibition of porin phenocopies Parkinson disease phenotypes in Drosophila, while the overexpression of Buffy can counteract these phenotypes to improve the overall "healthspan" of the organism.
Merzetti, E.M., Dolomount, L.A. and Staveley, B.E. (2016). The FBXO7 homologue nutcracker and binding partner PI31 in Drosophila melanogaster models of Parkinson's disease. Genome [Epub ahead of print]. PubMed ID: 27936908
Parkinsonian-pyramidal syndrome (PPS) is an early onset form of Parkinson's disease (PD) that shows degeneration of the extrapyramidal region of the brain to result in a severe form of PD. The toxic protein build-up has been implicated in the onset of PPS. Protein removal is mediated by an intracellular proteasome complex: an E3 ubiquitin ligase, the targeting component, is essential for function. FBXO7 encodes the F-box component of the SCF E3 ubiquitin ligase linked to familial forms of PPS. The Drosophila melanogaster homologue nutcracker (ntc) and a binding partner, PI31, have been shown to be active in proteasome function. This study shows that altered expression of either ntc or PI31 in dopaminergic neurons leads to a decrease in longevity and locomotor ability, phenotypes both associated with models of PD. Furthermore, expression of ntc-RNAi in an established α-synuclein-dependent model of PD rescues the phenotypes of diminished longevity and locomotor control.

Hindle, S. J., Hebbar, S., Schwudke, D., Elliott, C. J. and Sweeney, S. T. (2016). A saposin deficiency model in Drosophila: Lysosomal storage, progressive neurodegeneration and sensory physiological decline. Neurobiol Dis. PubMed ID: 27913291
Saposin deficiency is a childhood neurodegenerative lysosomal storage disorder (LSD) that can cause premature death within three months of life. Saposins are activator proteins that promote the function of lysosomal hydrolases that mediate the degradation of sphingolipids. Mutations causing an absence or impaired function of saposins in humans lead to distinct LSDs due to the storage of different classes of sphingolipids. The pathological events leading to neuronal dysfunction induced by lysosomal storage of sphingolipids are as yet poorly defined. A Drosophila model of saposin deficiency has been generated and characterised that shows striking similarities to the human diseases. Drosophila Saposin-related (dSap-r) mutants show a reduced longevity, progressive neurodegeneration, lysosomal storage, dramatic swelling of neuronal soma, perturbations in sphingolipid catabolism, and sensory physiological deterioration. These data suggests a genetic interaction with a calcium exchanger (Calx) pointing to a possible calcium homeostasis deficit in dSap-r mutants. Together these findings support the use of dSap-r mutants in advancing understanding of the cellular pathology implicated in saposin deficiency and related LSDs.

Saturday, December 24th

Diaz-Garcia, S., Ahmed, S. and Baonza, A. (2016). Analysis of the function of apoptosis during imaginal wing disc regeneration in Drosophila melanogaster. PLoS One 11(11): e0165554. PubMed ID: 27893747
Regeneration is the ability that allows organisms to replace missing organs or lost tissue after injuries. This ability requires the coordinated activity of different cellular processes, including programmed cell death. Apoptosis plays a key role as a source of signals necessary for regeneration in different organisms. The imaginal discs of Drosophila provide a particularly well-characterised model system for studying the cellular and molecular mechanisms underlying regeneration. Although it has been shown that signals produced by apoptotic cells are needed for homeostasis and regeneration of some tissues of this organism, such as the adult midgut, the contribution of apoptosis to disc regeneration remains unclear. Using a new method for studying disc regeneration in physiological conditions, this study has defined the pattern of cell death in regenerating discs. The data indicate that during disc regeneration, cell death increases first at the wound edge, but as regeneration progresses dead cells can be observed in regions far away from the site of damage. This result indicates that apoptotic signals initiated in the wound spread throughout the disc. Results are presented that suggest that the partial inhibition of apoptosis does not have a major effect on disc regeneration. Finally, these results suggest that during disc regeneration distinct apoptotic signals might be acting simultaneously.
M'Angale, P. G. and Staveley, B. E. (2016). Inhibition of Atg6 and Pi3K59F autophagy genes in neurons decreases lifespan and locomotor ability in Drosophila melanogaster.. Genet Mol Res 15. PubMed ID: 27813607
Autophagy is a cellular mechanism implicated in the pathology of Parkinson's disease. The proteins Atg6 (Beclin 1) and Pi3K59F are involved in autophagosome formation, a key step in the initiation of autophagy. This study used the GMR-Gal4 driver to determine the effect of reducing the expression of the genes encoding these proteins on the developing Drosophila eye. Subsequently, their expression in D. melanogaster neurons was inhibited under the direction of a Dopa decarboxylase (Ddc) transgene, and the effects on longevity and motor function were examined. Decreased longevity coupled with an age-dependent loss of climbing ability was observed. In addition, the roles of these genes were investigated in the well-studied alpha-synuclein-induced Drosophila model of Parkinson's disease. In this context, lowered expression of Atg6 or Pi3K59F in Ddc-Gal4-expressing neurons results in decreased longevity and associated age-dependent loss of locomotor ability. Inhibition of Atg6 or Pi3K59F together with overexpression of the sole pro-survival Bcl-2 Drosophila homolog Buffy in Ddc-Gal4-expressing neurons resulted in further decrease in the survival and climbing ability of Atg6-RNAi flies, whereas these measures were ameliorated in Pi3K59F-RNAi flies.
Varga, K., Nagy, P., Arsikin Csordas, K., Kovacs, A. L., Hegedus, K. and Juhasz, G. (2016). Loss of Atg16 delays the alcohol-induced sedation response via regulation of Corazonin neuropeptide production in Drosophila. Sci Rep 6: 34641. PubMed ID: 27708416
Autophagy defects lead to the buildup of damaged proteins and organelles, reduced survival during starvation and infections, hypersensitivity to stress and toxic substances, and progressive neurodegeneration. This study shows that, surprisingly, Drosophila mutants lacking the core autophagy gene Atg16 are not only defective in autophagy but also exhibit increased resistance to the sedative effects of ethanol, unlike Atg7 or Atg3 null mutant flies. This mutant phenotype is rescued by the re-expression of Atg16 in Corazonin (Crz)-producing neurosecretory cells that are known to promote the sedation response during ethanol exposure, and RNAi knockdown of Atg16 specifically in these cells also delays the onset of ethanol-induced coma. Atg16 and Crz colocalize within these neurosecretory cells, and both Crz protein and mRNA levels are decreased in Atg16 mutant flies. Thus, Atg16 promotes Crz production to ensure a proper organismal sedation response to ethanol.
Zhang, Y., Cui, C. and Lai, Z. C. (2016). The defender against apoptotic cell death 1 gene is required for tissue growth and efficient N-glycosylation in Drosophila melanogaster. Dev Biol [Epub ahead of print]. PubMed ID: 27693235
How organ growth is regulated in multicellular organisms is a long-standing question in developmental biology. It is known that coordination of cell apoptosis and proliferation is critical in cell number and overall organ size control, while how these processes are regulated is still under investigation. This study found that functional loss of a gene in Drosophila, named Drosophila defender against apoptotic cell death 1 (dDad1), leads to a reduction of tissue growth due to increased apoptosis and lack of cell proliferation. The dDad1 protein, an orthologue of mammalian Dad1, was found to be crucial for protein N-glycosylation in developing tissues. Loss of dDad1 function activates JNK signaling and blocking the JNK pathway in dDad1 knock-down tissues suppresses cell apoptosis and partially restores organ size. In addition, reduction of dDad1 triggers ER stress and activates unfolded protein response (UPR) signaling, prior to the activation of JNK signaling. Furthermore, Perk-Atf4 signaling, one branch of UPR pathways, appears to play a dual role in inducing cell apoptosis and mediating compensatory cell proliferation in this dDad1 knock-down model.

Friday, December 23rd

Jegla, T., et al. (2016). Bilaterian giant Ankyrins have a common evolutionary origin and play a conserved role in patterning the axon initial segment. PLoS Genet 12(12): e1006457. PubMed ID: 27911898
In vertebrate neurons, the axon initial segment (AIS) is specialized for action potential initiation. It is organized by a giant 480 Kd variant of ankyrin G (AnkG) that serves as an anchor for ion channels and is required for a plasma membrane diffusion barrier that excludes somatodendritic proteins from the axon. An unusually long exon required to encode this 480Kd variant is thought to have been inserted only recently during vertebrate evolution, so the giant ankyrin-based AIS scaffold has been viewed as a vertebrate adaptation for fast, precise signaling. This study re-examined AIS evolution through phylogenomic analysis of ankyrins and by testing the role of ankyrins in proximal axon organization in a model multipolar Drosophila neuron (ddaE). Giant isoforms of ankyrin were found in all major bilaterian phyla, and evidence is presented in favor of a single common origin for giant ankyrins and the corresponding long exon in a bilaterian ancestor. This finding raises the question of whether giant ankyrin isoforms play a conserved role in AIS organization throughout the Bilateria. This possibility was examined by looking for conserved ankyrin-dependent AIS features in Drosophila ddaE neurons via live imaging. ddaE neurons were found to have an axonal diffusion barrier proximal to the cell body that requires a giant isoform of the neuronal ankyrin Ank2. Furthermore, the potassium channel shal concentrates in the proximal axon in an Ank2-dependent manner. These results indicate that the giant ankyrin-based cytoskeleton of the AIS may have evolved prior to the radiation of extant bilaterian lineages, much earlier than previously thought.
Roh, S., Yang, D. S. and Jeong, S. (2016). Differential ligand regulation of PlexB signaling in motor neuron axon guidance in Drosophila. Int J Dev Neurosci 55: 34-40. PubMed ID: 27637927
Plexins (Plexs) are a large family of phylogenetically conserved guidance receptors that bind specifically to Semaphorins (Semas), another large family of guidance molecules. In the Drosophila embryonic central nervous system (CNS), the secreted semaphorins Sema-2a and Sema-2b both act as ligands for PlexB, but mediate mutually independent and opposite functions (repulsive and attractive guidance, respectively). PlexB is also known to regulate motor axon guidance in the embryonic peripheral nervous system (PNS). However, it is unclear whether the mechanisms of ligand regulation of PlexB seen in the CNS are similar or the same as those that exist in PNS motor axon guidance. This study finds that two distinct modes of ligand regulation underlie differential roles of PlexB in PNS motor axon pathfinding during embryonic development. Epistasis analyses in the intersegmental nerve b (ISNb) pathway suggest that PlexB serves as a receptor for both Sema-2a and Sema-2b and integrates their mutually dependent but opposite guidance functions. Furthermore, evidence is presented that PlexB mediates not only Sema-2a/2b-dependent guidance functions, but also Sema-2a/2b-independent target recognition in establishing the segmental nerve a (SNa) motor axon pathway. These results demonstrate that a single guidance receptor can elicit diverse effects on the establishment of neuronal connectivity via regulation of its ligands themselves.
Pearson, J.C., McKay, D.J., Lieb, J.D. and Crews, S.T. (2016). Chromatin profiling of Drosophila CNS subpopulations identifies active transcriptional enhancers. Development 143: 3723-3732. PubMed ID: 27802137
One of the key issues in studying transcriptional regulation during development is how to employ genome-wide assays that reveals sites of open chromatin and transcription factor binding to efficiently identify biologically relevant genes and enhancers. Analysis of Drosophila CNS midline cell development provides a useful system for studying transcriptional regulation at the genomic level due to a large, well-characterized set of midline-expressed genes and in vivo validated enhancers. In this study, Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE-seq) was performed on FACS-purified midline cells and the midline FAIRE data were compared with whole-embryo FAIRE data. It was found that regions of the genome with a strong midline FAIRE peak and weak whole-embryo FAIRE peak overlap with known midline enhancers and provide a useful predictive tool for enhancer identification. In a complementary analysis, a large dataset of fragments that drive midline expression in vivo was compared with the FAIRE data. Midline enhancer fragments with a midline FAIRE peak tend to be near midline-expressed genes, whereas midline enhancers without a midline FAIRE peak are often distant from midline-expressed genes and unlikely to drive midline transcription in vivo.

Hessinger, C., Technau, G.M. and Rogulja-Ortmann, A. (2016). The Drosophila Hox gene Ultrabithorax acts both in muscles and motoneurons to orchestrate formation of specific neuromuscular connections. Development [Epub ahead of print]. PubMed ID: 27913640
Hox genes are known to specify motoneuron pools in the developing vertebrate spinal cord and to control motoneuronal targeting in several species. However, the mechanisms controlling axial diversification of muscle innervation patterns are still largely unknown. This study presents data showing that the Drosophila Hox gene Ultrabithorax (Ubx) acts in the late embryo to establish target specificity of ventrally projecting RP motoneurons. In abdominal segments A2 to A7, RP motoneurons innervate the ventro-lateral muscles VL1-4, with VL1 and VL2 being innervated in a Wnt4-dependent manner. In Ubx mutants, these motoneurons fail to make correct contacts with muscle VL1, a phenotype partially resembling that of the Wnt4 mutant. Ubx regulates expression of Wnt4 in muscle VL2 and interacts with the Wnt4 response pathway in the respective motoneurons. Ubx thus orchestrates the interaction between two cell types, muscles and motoneurons, to regulate establishment of the ventro-lateral neuromuscular network.

Thursday, December 22nd

Li, Y., Dharkar, P., Han, T. H., Serpe, M., Lee, C. H. and Mayer, M. L. (2016). Novel functional properties of Drosophila CNS glutamate receptors. Neuron 92(5): 1036-1048. PubMed ID: 27889096
Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation. See the Video Abstract.
Guntur, A. R., Gou, B., Gu, P., He, R., Stern, U., Xiang, Y. and Yang, C. H. (2016). H2O2-sensitive isoforms of Drosophila TRPA1 act in bitter-sensing gustatory neurons to promote avoidance of UV during egg-laying. Genetics [Epub ahead of print]. PubMed ID: 27932542
Recent results have suggested that specific isoforms of Drosophila TRPA1 (dTRPA1) are UV sensitive and that their UV sensitivity is due to H2O2 sensitivity. This study has demonstrated that H2O2-sensitive dTRPA1 isoforms promote avoidance of UV when adult Drosophila females are selecting sites for egg-laying. Blind/visionless females are still capable of sensing and avoiding UV during egg-laying when intensity of UV is high yet within the range of natural sunlight. Second, such vision-independent UV avoidance was shown to be mediated by a group of bitter-sensing neurons on the proboscis that express H2O2-sensitive dTRPA1 isoforms. These bitter-sensing neurons exhibit dTRPA1-dependent UV sensitivity. Importantly, inhibiting activities of these bitter-sensing neurons, reducing their dTRPA1 expression, or reducing their H2O2-sensitivity all significantly reduced blind females' UV avoidance, whereas selectively restoring a H2O2-sensitive isoform of dTRPA1 in these neurons restored UV avoidance. Lastly, it was shown that expressing the red-shifted channelrhodopsin CsChrimson specifically in these bitter-sensing neurons promotes egg-laying avoidance of red light, an otherwise neutral cue for egg-laying females.
Cervantes, S. A., Bajakian, T. H., Soria, M. A., Falk, A. S., Service, R. J., Langen, R. and Siemer, A. B. (2016). Identification and structural characterization of the N-terminal amyloid core of Orb2 isoform A. Sci Rep 6: 38265. PubMed ID: 27922050
Orb2 is a functional amyloid that plays a key role in Drosophila long-term memory formation. Orb2 has two isoforms that differ in their N-termini. The N-terminus of the A isoform (Orb2A) that precedes its Q-rich prion-like domain has been shown to be important for Orb2 aggregation and long-term memory. However, besides the fact that it forms fibrillar aggregates, structural information of Orb2 is largely absent. To understand the importance of the N-terminus of Orb2A and its relation to the fibril core, solid-state NMR and EPR data were recorded on fibrils formed by the first 88 residues of Orb2A (Orb2A88). These data show that the N-terminus of Orb2A not only promotes the formation of fibrils, but also forms the fibril core of Orb2A88. This fibril core has an in-register parallel beta-sheet structure and does not include the Q-rich, prion-like domain of Orb2. The Q-rich domain is part of the unstructured region, which becomes increasingly dynamic towards the C-terminus.
Chen, X., Rahman, R., Guo, F. and Rosbash, M. (2016). Genome-wide identification of neuronal activity-regulated genes in Drosophila. Elife 5. PubMed ID: 27936378
Activity-regulated genes (ARGs) are important for neuronal functions like long-term memory and are well-characterized in mammals but poorly studied in other model organisms like Drosophila. This study stimulated fly neurons with different paradigms and identified ARGs using high-throughput sequencing from brains as well as from sorted neurons: they included a narrow set of circadian neurons as well as dopaminergic neurons. Surprisingly, many ARGs are specific to the stimulation paradigm and very specific to neuron type. In addition and unlike mammalian immediate early genes (IEGs), fly ARGs, including hr38, stripe, ring finger protein CG8910, and the protein kinase CG11221, do not have short gene lengths and are less enriched for transcription factor function. Chromatin assays using ATAC-sequencing show that the transcription start sites (TSS) of ARGs do not change with neural firing but are already accessible prior to stimulation. Lastly based on binding site enrichment in ARGs, transcription factor mediators of firing were identified, and neuronal activity reporters were created.

Wednesday, December 21st

Kandasamy, S. K. and Fukunaga, R. (2016). Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production. Proc Natl Acad Sci U S A 113(49): 14031-14036. PubMed ID: 27872309
The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) . In Drosophila, Dicer-1 produces approximately 22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. This study reports that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. It is proposed that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.
Haussmann, I. U., Bodi, Z., Sanchez-Moran, E., Mongan, N. P., Archer, N., Fray, R. G. and Soller, M. (2016). m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination. Nature 540(7632): 301-304. PubMed ID: 27919081
N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. Drosophila mRNA m6A methylosome consists of Ime4 and KAR4 (Inducer of meiosis 4 and Karyogamy protein 4), and Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain. This study shows that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout models, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. The m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression.
Dennis, C., Brasset, E., Sarkar, A. and Vaury, C. (2016). Export of piRNA precursors by EJC triggers assembly of cytoplasmic Yb-body in Drosophila. Nat Commun 7: 13739. PubMed ID: 27929060
PIWI-interacting RNAs (piRNAs) are effectors of transposable element (TE) silencing in the reproductive apparatus. In Drosophila ovarian somatic cells, piRNAs arise from longer single-stranded RNA precursors that are processed in the cytoplasm presumably within the Yb-bodies. piRNA precursors encoded by the flamenco (flam) piRNA cluster accumulate in a single focus away from their sites of transcription. This study identifies the exportin complex containing Nxf1 and Nxt1 as being required for flam precursor nuclear export. Together with components of the exon junction complex (EJC), it is necessary for the efficient transfer of flam precursors away from their site of transcription. Indeed, depletion of these components greatly affects flam intra-nuclear transit. Moreover, Yb-body assembly is dependent on the nucleo-cytoplasmic export of flam transcripts. These results suggest that somatic piRNA precursors are required for the assembly of the cytoplasmic transposon silencing machinery.

Gibilisco, L., Zhou, Q., Mahajan, S. and Bachtrog, D. (2016). Alternative splicing within and between Drosophila species, sexes, tissues, and developmental stages. PLoS Genet 12(12): e1006464. PubMed ID: 27935948
Alternative pre-mRNA splicing ("AS") greatly expands proteome diversity. The transcriptomes from several tissues and developmental stages were studied in males and females from four species across the Drosophila genus. 20-37% of multi-exon genes were found to be alternatively spliced. While males generally express a larger number of genes, AS is more prevalent in females, suggesting that the sexes adopt different expression strategies for their specialized function. The proportion of expressed genes that are alternatively spliced is highest in the very early embryo, before the onset of zygotic transcription. This indicates that females deposit a diversity of isoforms into the egg, consistent with abundant AS found in ovary. Cluster analysis by gene expression levels shows mostly stage-specific clustering in embryonic samples, and tissue-specific clustering in adult tissues. Clustering embryonic stages and adult tissues based on AS profiles results in stronger species-specific clustering, suggesting that diversification of splicing contributes to lineage-specific evolution in Drosophila. Most sex-biased AS found in flies is due to AS in gonads, with little sex-specific splicing in somatic tissues.

Tuesday, December 20th

Chen, L., Nye, D.M., Stone, M.C., Weiner, A.T., Gheres, K.W., Xiong, X., Collins, C.A. and Rolls, M.M. (2016). Mitochondria and caspases tune Nmnat-mediated stabilization to promote axon regeneration. PLoS Genet 12: e1006503. PubMed ID: 27923046
Axon injury can lead to several cell survival responses including increased stability and axon regeneration. Using an accessible Drosophila model system, this study investigated the regulation of injury responses and their relationship. Axon injury stabilizes the rest of the cell, including the entire dendrite arbor. After axon injury, it was found that mitochondrial fission in dendrites is upregulated, and reducing fission increases stabilization or neuroprotection (NP). Thus axon injury seems to both turn on NP, but also dampen it by activating mitochondrial fission. Caspases were identified to be negative regulators of axon injury-mediated NP, so mitochondrial fission could control NP through caspase activation. In addition to negative regulators of NP, it was found that nicotinamide mononucleotide adenylyltransferase (Nmnat) is absolutely required for this type of NP. Increased microtubule dynamics, which has previously been associated with NP, requires Nmnat. Indeed Nmnat overexpression is sufficient to induce NP and increase microtubule dynamics in the absence of axon injury. DLK, JNK and fos are also required for NP. Because NP occurs before axon regeneration, and NP seems to be actively downregulated, it was tested whether excessive NP might inhibit regeneration. Indeed both Nmnat overexpression and caspase reduction reduce regeneration. In addition, overexpression of fos or JNK extend the timecourse of NP and dampen regeneration in a Nmnat-dependent manner. These data suggest that NP and regeneration are conflicting responses to axon injury, and that therapeutic strategies that boost NP may reduce regeneration.

Munkácsy, E., Khan, M.H., Lane, R.K., Borror, M.B., Park, J.H., Bokov, A.F., Fisher, A.L., Link, C.D. and Rea, S.L. (2016). DLK-1, SEK-3 and PMK-3 are required for the life extension induced by mitochondrial bioenergetic disruption in C. elegans. PLoS Genet 12: e1006133. PubMed ID: 27420916
Evolutionary Homolog Study:
Mitochondrial dysfunction underlies numerous age-related pathologies. In an effort to uncover how the detrimental effects of mitochondrial dysfunction might be alleviated, this study examined how the nematode C. elegans not only adapts to disruption of the mitochondrial electron transport chain, but in many instances responds with extended lifespan. Studies have shown various retrograde responses are activated in these animals, including the well-studied ATFS-1-dependent mitochondrial unfolded protein response (UPRmt) (see Drosophila crc and UPR). Such processes fall under the greater rubric of cellular surveillance mechanisms. This study identified a novel p38 signaling cascade that is required to extend life in worms upon disruption of the mitochondrial electron transport chain, and which is blocked by disruption of the Mitochondrial-associated Degradation (MAD) pathway. This novel cascade is defined by DLK-1 (MAP3K) (see Drosophila wnd), SEK-3 (MAP2K) (see Drosophila Mkk4), PMK-3 (MAPK) (see Drosophila p38a) and the reporter gene Ptbb-6::GFP. Inhibition of known mitochondrial retrograde responses does not alter induction of Ptbb-6::GFP, instead induction of this reporter often occurs in counterpoint to activation of SKN-1, which was shown to be under the control of ATFS-1. In those mitochondrial bioenergetic mutants which activate Ptbb-6::GFP, it was found that dlk-1, sek-3 and pmk-3 are all required for their life extension.

Rauschenbach, I. Y., Karpova, E. K., Burdina, E. V., Adonyeva, N. V., Bykov, R. A., Ilinsky, Y. Y., Menshanov, P. N. and Gruntenko, N. E. (2016). Insulin-like peptide DILP6 regulates juvenile hormone and dopamine metabolism in Drosophila females. Gen Comp Endocrinol 243: 1-9. PubMed ID: 27823956
Insulin-like peptide DILP6 is a component of the insulin/insulin-like growth factor signalling pathway of Drosophila. Juvenile hormone (JH) and dopamine (DA) are involved in the stress response and in the control of reproduction. This study investigated whether DILP6 regulates the JH and DA levels by studying the effect of a strong hypomorphic mutation dilp641 on JH and DA metabolism in D. melanogaster females. DILP6 was shown to regulate JH and DA metabolism: the mutation dilp641 results in a reduction in JH-hydrolysing activity and an increase in the activities of DA synthesis enzymes (alkaline phosphatase (ALP) and tyrosine hydroxylase (TH)). In the mutant females, increased fecundity was found, in addition to the intensity of the response (stress reactivity) of ALP and TH to heat stress. This suggests an increased level of JH synthesis. This suggestion was confirmed by treating the mutant females with the JH inhibitor, precocene, which restores the activity and stress reactivity of ALP and TH as well as fecundity to levels similar to those in the control flies. The data suggest a feedback system in the interaction between JH and DILP6 in which DILP6 negatively regulates the JH titre via an increase in the hormone degradation and a decrease in its synthesis.
Xie, G., Chen, H., Jia, D., Shu, Z., Palmer, W.H., Huang, Y.C., Zeng, X., Hou, S.X., Jiao, R.2 and Deng, W.M. (2016). The SWI/SNF complex protein Snr1 is a tumor suppressor in Drosophila imaginal tissues. Cancer Res [Epub ahead of print]. PubMed ID: 27923836
Components of the SWI/SNF chromatin-remodeling complex are among the most frequently mutated genes in various human cancers, yet only SMARCB1/hSNF5, a core member of the SWI/SNF complex, is mutated in malignant rhabdoid tumors (MRT). How SMARCB1/hSNF5 functions differently from other members of the SWI/SNF complex remains unclear. This study used Drosophila imaginal epithelial tissues to demonstrate that Snr1, the conserved homolog of human SMARCB1/hSNF5, prevents tumorigenesis by maintaining normal endosomal trafficking-mediated signaling cascades. Removal of Snr1 results in neoplastic tumorigenic overgrowth in imaginal epithelial tissues, whereas depletion of any other members of the SWI/SNF complex does not induce similar phenotypes. Unlike other components of the SWI/SNF complex that are detected only in the nucleus, Snr1 is observed in both the nucleus and the cytoplasm. Aberrant regulation of multiple signaling pathways including Notch, JNK, and JAK/STAT, is responsible for tumor progression upon Snr1-depletion. These results suggest that the cytoplasmic Snr1 may play a tumor suppressive role in Drosophila imaginal tissues, offering a foundation for understanding the pivotal role of SMARCB1 in suppressing MRT during early childhood.

Hallier, B., Schiemann, R., Cordes, E., Vitos-Faleato, J., Walter, S., Heinisch, J. J., Malmendal, A., Paululat, A. and Meyer, H. (2016). Drosophila neprilysins control insulin signaling and food intake via cleavage of regulatory peptides. Elife 5. PubMed ID: 27919317
Insulin and IGF signaling are critical to numerous developmental and physiological processes, with perturbations being pathognomonic of various diseases, including diabetes. Although the functional roles of the respective signaling pathways have been extensively studied, the control of insulin production and release is only partially understood. This study shows that in Drosophila expression of insulin-like peptides is regulated by neprilysin activity. Concomitant phenotypes of altered expression of the metallopeptidase neprilysin (see Neprilysin 4), included impaired food intake, reduced body size, and characteristic changes in the metabolite composition. Ectopic expression of a catalytically inactive mutant did not elicit any of the phenotypes, which confirms abnormal peptide hydrolysis as a causative factor. A screen for corresponding substrates of the neprilysin identified distinct peptides that regulate insulin-like peptide expression, feeding behavior, or both. The high functional conservation of neprilysins and their substrates renders the characterized principles applicable to numerous species, including higher eukaryotes and humans.
Boone, E., Colombani, J., Andersen, D. S. and Leopold, P. (2016). The Hippo signalling pathway coordinates organ growth and limits developmental variability by controlling dilp8 expression. Nat Commun 7: 13505. PubMed ID: 27874005
Coordination of organ growth during development is required to generate fit individuals with fixed proportions. Dilp8 has been identified as a key hormone in coupling organ growth with animal maturation. In addition, dilp8 mutant flies exhibit elevated fluctuating asymmetry (FA) demonstrating a function for Dilp8 in ensuring developmental stability. The signals regulating Dilp8 activity during normal development are not yet known. This study shows that the transcriptional co-activators of the Hippo (Hpo) pathway, Yorkie (Yki, YAP/TAZ) and its DNA-binding partner Scalloped (Sd), directly regulate dilp8 expression through a Hpo-responsive element (HRE) in the dilp8 promoter. It was further demonstrated that mutation of the HRE by genome-editing results in animals with increased FA, thereby mimicking full dilp8 loss of function. Therefore, these results indicate that growth coordination of organs is connected to their growth status through a feedback loop involving Hpo and Dilp8 signalling pathways.
Chen YC, Chen HJ, Tseng WC, Hsu JM, Huang TT, Chen CH, Pan CL. (2016).. A C. elegans thermosensory circuit regulates longevity through crh-1/CREB-dependent flp-6 neuropeptide signaling. JDev Cell 39(2):209-223. PubMed ID: 27720609
Evolutionary Homolog Study
Sensory perception, including thermosensation, shapes longevity in diverse organisms, but longevity-modulating signals from the sensory neurons are largely obscure. This study shows that CRH-1/CREB (see Drosophila CrebB) activation by CMK-1/CaMKI in the AFD thermosensory neuron is a key mechanism that maintains lifespan at warm temperatures in C. elegans. In response to temperature rise and crh-1 activation, the AFD neurons produce and secrete the FMRFamide neuropeptide FLP-6. Both CRH-1 and FLP-6 are necessary and sufficient for longevity at warm temperatures. These data suggest that FLP-6 targets the AIY interneurons and engages DAF-9 sterol hormone signaling. Moreover, it was shown that FLP-6 signaling downregulates ins-7/insulin-like peptide and several insulin pathway genes, whose activity compromises lifespan. This work illustrates how temperature experience is integrated by the thermosensory circuit to generate neuropeptide signals that remodel insulin and sterol hormone signaling and reveals a neuronal-endocrine circuit driven by thermosensation to promote temperature-specific longevity.
Kaplan, M., Narasimhan, S., de Heus, C., Mance, D., van Doorn, S., Houben, K., Popov-Celeketic, D., Damman, R., Katrukha, E. A., Jain, P., Geerts, W. J., Heck, A. J., Folkers, G. E., Kapitein, L. C., Lemeer, S., van Bergen En Henegouwen, P. M. and Baldus, M. (2016). EGFR dynamics change during activation in native membranes as revealed by NMR. Cell 167(5): 1241-1251. PubMed ID: 27839865
Evolutionary Homolog Study
The epidermal growth factor receptor (EGFR; see Drosophila Egfr) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP) was developed. In contrast to previous crystallographic results, the current experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. It is proposes that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.

Monday, December 19th

Cubenas-Potts, C., Rowley, M. J., Lyu, X., Li, G., Lei, E. P. and Corces, V. G. (2016). Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 27899590
Genome-wide studies has identified two enhancer classes in Drosophila that interact with different core promoters: housekeeping enhancers (hkCP) and developmental enhancers (dCP). It is hypothesized that the two enhancer classes are occupied by distinct architectural proteins, affecting their enhancer-promoter contacts. It was determined that both enhancer classes are enriched for RNA Polymerase II, CBP, and architectural proteins but there are also distinctions. hkCP enhancers contain H3K4me3 and exclusively bind Cap-H2, Chromator, DREF and Z4, whereas dCP enhancers contain H3K4me1 and are more enriched for Rad21 and Fs(1)h-L. Additionally, the interactions of each enhancer class were mapped utilizing a Hi-C dataset with <1 kb resolution. Results suggest that hkCP enhancers are more likely to form multi-TSS interaction networks and be associated with topologically associating domain (TAD) borders, while dCP enhancers are more often bound to one or two TSSs and are enriched at chromatin loop anchors. The data support a model suggesting that the unique architectural protein occupancy within enhancers is one contributor to enhancer-promoter interaction specificity.
Ali, T., Krüger, M., Bhuju, S., Jarek, M., Bartkuhn, M. and Renkawitz, R. (2016). Chromatin binding of Gcn5 in Drosophila is largely mediated by CP190. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 27903907
Centrosomal 190 kDa protein (CP190) is a promoter binding factor, mediates long-range interactions in the context of enhancer-promoter contacts and in chromosomal domain formation. All Drosophila insulator proteins bind CP190 suggesting a crucial role in insulator function. CP190 has major effects on chromatin, such as depletion of nucleosomes, high nucleosomal turnover and prevention of heterochromatin expansion. This study searched for enzymes which might be involved in CP190 mediated chromatin changes. Eighty percent of the genomic binding sites of the histone acetyltransferase Gcn5 colocalize with CP190 binding. Depletion of CP190 reduces Gcn5 binding to chromatin. Binding dependency is further supported by Gcn5 mediated co-precipitation of CP190. Gcn5 is known to activate transcription by histone acetylation. The dCas9 system was used to target CP190 or Gcn5 to a Polycomb repressed and H3K27me3 marked gene locus; both CP190 as well as Gcn5 activate this locus, thus supporting the model that CP190 recruits Gcn5 and thereby activates chromatin.

Blythe, S. A. and Wieschaus, E. F. (2016). Establishment and maintenance of heritable chromatin structure during early Drosophila embryogenesis. Elife 5. PubMed ID: 27879204
During embryogenesis, the initial chromatin state is established during a period of rapid proliferative activity. This study measured with three-minute time resolution how heritable patterns of chromatin structure are initially established and maintained during the midblastula transition (MBT). Regions of accessibility are established sequentially, where enhancers are opened in advance of promoters and insulators. These open states are stably maintained in highly condensed mitotic chromatin to ensure faithful inheritance of prior accessibility status across cell divisions. The temporal progression of establishment is controlled by the biological timers that control the onset of the MBT. In general, acquisition of promoter accessibility is controlled by the biological timer that measures the nucleo-cytoplasmic (N:C) ratio whereas timing of enhancer accessibility is regulated independently of the N:C ratio. These different timing classes each associate with binding sites for two transcription factors, GAGA-factor and Zelda, previously implicated in controlling chromatin accessibility at ZGA.
Lopez-Mosqueda, J., Maddi, K., Prgomet, S., Kalayil, S., Marinovic-Terzic, I., Terzic, J. and Dikic, I. (2016). SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks. Elife 5. PubMed ID: 27852435
Evolutionary Homolog Study:
Ruijs-Aalfs syndrome is a segmental progeroid syndrome resulting from mutations in the SPRTN (see Drosophila maternal haploid) gene. Cells derived from patients with SPRTN mutations elicit genomic instability and people afflicted with this syndrome develop hepatocellular carcinoma. This study describes the molecular mechanism by which SPRTN contributes to genome stability and normal cellular homeostasis. It was shown that SPRTN is a DNA-dependent mammalian protease required for resolving cytotoxic DNA-protein crosslinks (DPCs)- a function that had only been attributed to the metalloprotease Wss1 in budding yeast. The study provides genetic evidence that SPRTN and Wss1 function distinctly in vivo to resolve DPCs. Upon DNA and ubiquitin binding, SPRTN can elicit proteolytic activity; cleaving DPC substrates and itself. SPRTN null cells or cells derived from patients with Ruijs-Aalfs syndrome are impaired in the resolution of covalent DPCs in vivo. Collectively, SPRTN is a mammalian protease required for resolving DNA-protein crosslinks in vivo whose function is compromised in Ruijs-Aalfs syndrome patients.

Sunday, December 18th

Zimmerman, J.E., Chan, M.T., Lenz, O.T., Keenan, B.T., Maislin, G. and Pack, A.I. (2016). Glutamate is a wake active neurotransmitter in Drosophila melanogaster. Sleep [Epub ahead of print]. PubMed ID: 27855736
In mammals, there is evidence that glutamate has a role as a wake active neurotransmitter. This study examined if glutamate, which has been shown to have an excitatory role in neuromuscular junctions in Drosophila, has a wake-active role in the adult brain. Using 6-9 day old female flies, the effect of perturbations of the glutamatergic signaling on total wakefulness and wake bout architecture were examined. The neuronal activity of glutamatergic neurons in the brains of adult flies were increased and decreased using UAS NaChBac and UAS EKO, respectively. Neurotransmission was blocked from glutamatergic neurons in adult flies using the UAS driven temperature sensitive dynamin mutation shibirets. Increasing the activity of glutamatergic neurons in the adult brain leads to a significant increase in wakefulness both in the daytime and nighttime and decreasing the activity of these same neurons reduces wakefulness in the nighttime. Blocking neurotransmitter release in glutamatergic neurons significantly reduces wake in the nighttime. The results show that: glutamate is indeed a wake-active neurotransmitter in Drosophila; there is a major time of day effect associated with loss of glutamatergic neurotransmission; and it is a major wake-active neurotransmitter in the nighttime.

Murphy, K.R., Deshpande, S.A., Yurgel, M.E., Quinn, J.P., Weissbach, J.L., Keene, A.C., Dawson-Scully, K., Huber, R., Tomchik, S.M. and Ja, W.W. (2016). Postprandial sleep mechanics in Drosophila. Elife 5. PubMed ID: 27873574
Food consumption is thought to induce sleepiness. However, little is known about how postprandial sleep is regulated. This study simultaneously measured sleep and food intake of individual flies and found a transient rise in sleep following meals. Depending on the amount consumed, the effect ranges from slightly arousing to strongly sleep inducing. Postprandial sleep was positively correlated with ingested volume, protein, and salt-but not sucrose-revealing meal property-specific regulation. Silencing of leucokinin receptor (Lkr) neurons specifically reduces sleep induced by protein consumption. Thermogenetic stimulation of leucokinin (Lk) neurons decreases whereas Lk downregulation by RNAi increases postprandial sleep, suggestive of an inhibitory connection in the Lk-Lkr circuit. A subset of non-leucokininergic cells proximal to Lkr neurons were identified to rhythmically increase postprandial sleep when silenced, suggesting that these cells are cyclically gated inhibitory inputs to Lkr neurons. Together, these findings reveal the dynamic nature of postprandial sleep.

Cho, E., Oh, J. H., Lee, E., Do, Y. R. and Kim, E. Y. (2016). Cycles of circadian illuminance are sufficient to entrain and maintain circadian locomotor rhythms in Drosophila. Sci Rep 6: 37784. PubMed ID: 27883065
Light at night disrupts the circadian clock and causes serious health problems in the modern world. This study shows that newly developed four-package light-emitting diodes (LEDs) can provide harmless lighting at night. To quantify the effects of light on the circadian clock, the concept of circadian illuminance (CIL) was employed. CIL represents the amount of light weighted toward the wavelengths to which the circadian clock is most sensitive, whereas visual illuminance (VIL) represents the total amount of visible light. Exposure to 12 h:12 h cycles of white LED light with high and low CIL values but a constant VIL value (conditions hereafter referred to as CH/CL) can entrain behavioral and molecular circadian rhythms in flies. Moreover, flies re-entrain to phase shift in the CH/CL cycle. Core-clock proteins are required for the rhythmic behaviors seen with this LED lighting scheme. Taken together, this study provides a guide for designing healthful white LED lights for use at night, and proposes the use of the CIL value for estimating the harmful effects of any light source on organismal health.
Harper, R. E., Dayan, P., Albert, J. T. and Stanewsky, R. (2016). Sensory conflict disrupts activity of the Drosophila circadian network. Cell Rep 17: 1711-1718. PubMed ID: 27829142
Periodic changes in light and temperature synchronize the Drosophila circadian clock, but the question of how the fly brain integrates these two input pathways to set circadian time remains unanswered. This study explored multisensory cue combination by testing the resilience of the circadian network to conflicting environmental inputs. Misaligned light and temperature cycles can lead to dramatic changes in the daily locomotor activities of wild-type flies during and after exposure to sensory conflict. This altered behavior is associated with a drastic reduction in the amplitude of Period (Per) oscillations in brain clock neurons and desynchronization between light- and temperature-sensitive neuronal subgroups. The behavioral disruption depends heavily on the phase relationship between light and temperature signals. These results represent a systematic quantification of multisensory integration in the Drosophila circadian system and lend further support to the view of the clock as a network of coupled oscillatory subunits.

Saturday, December 17th

Lee, Y. C., Leek, C. and Levine, M. T. (2016). Recurrent innovation at genes required for telomere integrity in Drosophila. Mol Biol Evol [Epub ahead of print]. PubMed ID: 27836984
Telomeres are nucleoprotein complexes at the ends of linear chromosomes. These specialized structures ensure genome integrity and faithful chromosome inheritance. Recurrent addition of repetitive, telomere-specific DNA elements to chromosome ends combats end-attrition, while specialized telomere-associated proteins protect naked, double-stranded chromosome ends from promiscuous repair into end-to-end fusions. Although telomere length homeostasis and end-protection are ubiquitous across eukaryotes, there is sporadic but building evidence that the molecular machinery supporting these essential processes evolves rapidly. Nevertheless, no global analysis of the evolutionary forces that shape these fast-evolving proteins has been performed on any eukaryote. The abundant population and comparative genomic resources of Drosophila melanogaster and its close relatives offer a unique opportunity to fill this gap. This study leverages population genetics, molecular evolution, and phylogenomics to define the scope and evolutionary mechanisms driving fast evolution of genes required for telomere integrity. Evidence was uncovered of pervasive positive selection across multiple evolutionary timescales. Prolific expansion, turnover, and expression evolution was documented in gene families founded by telomeric proteins. Motivated by the mutant phenotypes and molecular roles of these fast-evolving genes, four alternative, but not mutually exclusive, models were proposed of intra-genomic conflict that may play out at very termini of eukaryotic chromosomes. The findings set the stage for investigating both the genetic causes and functional consequences of telomere protein evolution in Drosophila and beyond.
Pauli, T., et al. (2016). Transcriptomic data from panarthropods shed new light on the evolution of insulator binding proteins in insects. BMC Genomics 17: 861. PubMed ID: 27809783
Body plan development in multi-cellular organisms is largely determined by homeotic genes. Expression of homeotic genes, in turn, is partially regulated by insulator binding proteins (IBPs). While only a few enhancer blocking IBPs have been identified in vertebrates, the common fruit fly Drosophila melanogaster harbors at least twelve different enhancer blocking IBPs. This study screened ecently compiled insect transcriptomes from the 1KITE project and genomic and transcriptomic data from public databases, aiming to trace the origin of IBPs in insects and other arthropods. The study shows that the last common ancestor of insects (Hexapoda) already possessed a substantial number of IBPs. Specifically, of the known twelve insect IBPs, at least three (i.e., CP190, Su(Hw), and CTCF) already existed prior to the evolution of insects. Furthermore GAF orthologs were found in early branching insect orders, including Zygentoma (silverfish and firebrats) and Diplura (two-pronged bristletails). Mod(mdg4) is most likely a derived feature of Neoptera, while Pita is likely an evolutionary novelty of holometabolous insects. Zw5 appears to be restricted to schizophoran flies, whereas BEAF-32, ZIPIC and the Elba complex, are probably unique to the genus Drosophila. Selection models indicate that insect IBPs evolved under neutral or purifying selection. These results suggest that a substantial number of IBPs either pre-date the evolution of insects or evolved early during insect evolution. This suggests an evolutionary history of insulator binding proteins in insects different to that previously thought. Moreover, this study demonstrates the versatility of the 1KITE transcriptomic data for comparative analyses in insects and other arthropods.
Prieto-Godino, L. L., Rytz, R., Bargeton, B., Abuin, L., Arguello, J. R., Peraro, M. D. and Benton, R. (2016). Olfactory receptor pseudo-pseudogenes. Nature 539: 93-97. PubMed ID: 27776356
Pseudogenes are generally considered to be non-functional DNA sequences that arise through nonsense or frame-shift mutations of protein-coding genes. Although certain pseudogene-derived RNAs have regulatory roles, and some pseudogene fragments are translated, no clear functions for pseudogene-derived proteins are known. Olfactory receptor families contain many pseudogenes, which reflect low selection pressures on loci no longer relevant to the fitness of a species. This study reports the characterization of a pseudogene in the chemosensory variant ionotropic glutamate receptor repertoire of Drosophila sechellia, an insect endemic to the Seychelles that feeds almost exclusively on the ripe fruit of Morinda citrifolia. This locus, D. sechellia Ir75a (see Drosophila Ir75a), bears a premature termination codon (PTC) that appears to be fixed in the population. However, D. sechellia Ir75a encodes a functional receptor, owing to efficient translational read-through of the PTC. Read-through is detected only in neurons and is independent of the type of termination codon, but depends on the sequence downstream of the PTC. Furthermore, although the intact Drosophila melanogaster Ir75a orthologue detects acetic acid-a chemical cue important for locating fermenting food found only at trace levels in Morinda fruit-D. sechellia Ir75a has evolved distinct odour-tuning properties through amino-acid changes in its ligand-binding domain. Functional PTC-containing loci were identified within different olfactory receptor repertoires and species, suggesting that such 'pseudo-pseudogenes' could represent a widespread phenomenon.
Almeida, F. C. and DeSalle, R. (2016). Genetic differentiation and adaptive evolution at reproductive loci in incipient Drosophila species. J Evol Biol. PubMed ID: 27883252
Accessory gland proteins (Acps; see Drosophila Sex Peptide and 0vulin) are part of the seminal fluid of male Drosophila flies. Some Acps have exceptionally high evolutionary rates and evolve under positive selection. Proper interactions between Acps and female reproductive molecules are essential for fertilization. These observations lead to suggestions that fast evolving Acps could be involved in speciation by promoting reproductive incompatibilities between emerging species. To test this hypothesis, population genetics data were used for three sibling species: D. mayaguana, D. parisiena, and D. straubae. The latter two species are morphologically very similar and show only incipient reproductive isolation. This system allowed examination of Acp evolution at different time frames in respect to speciation and reproductive isolation. Comparing data of 14 Acp loci with data obtained for other genomic regions, it was found that some Acps show extraordinarily high levels of divergence between D. mayaguana and its two sister species D. parisiena, and D. straubae. This divergence was likely driven by adaptive evolution at several loci. No fixed nucleotide differences were found between D. parisiena and D. straubae, however. Nevertheless, some Acp loci did show significant differentiation between these species associated with signs of positive selection; these loci may be involved in this early phase of the speciation process.

Friday, December 16th

Ulmschneider, B., Grillo-Hill, B.K., Benitez, M., Azimova, D.R., Barber, D.L. and Nystul, T.G. (2016). Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation. J Cell Biol 215: 345-355. PubMed ID: 27821494
Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. This study reports that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). It was shown that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+-H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. These findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms.

Chen, C., Inaba, M., Venkei, Z. G. and Yamashita, Y. M. (2016). Klp10A, a stem cell centrosome-enriched kinesin, balances asymmetries in Drosophila male germline stem cell division. Elife 5. PubMed ID: 27885983
Asymmetric stem cell division is often accompanied by stereotypical inheritance of the mother and daughter centrosomes. However, it remains unknown whether and how stem cell centrosomes are uniquely regulated and how this regulation may contribute to stem cell fate. This study identifies Klp10A, a microtubule-depolymerizing kinesin of the kinesin-13 family, as the first protein enriched in the stem cell centrosome in Drosophila male germline stem cells (GSCs). Depletion of klp10A results in abnormal elongation of the mother centrosomes in GSCs, suggesting the existence of a stem cell-specific centrosome regulation program. Concomitant with mother centrosome elongation, GSCs form asymmetric spindle, wherein the elongated mother centrosome organizes considerably larger half spindle than the other. This leads to asymmetric cell size, yielding a smaller differentiating daughter cell. It is proposed that klp10A functions to counteract undesirable asymmetries that may result as a by-product of achieving asymmetries essential for successful stem cell divisions.
Bernitz, J. M., Kim, H. S., MacArthur, B., Sieburg, H. and Moore, K. (2016). Hematopoietic stem cells count and remember self-renewal divisions. Cell 167(5): 1296-1309 e1210. PubMed ID: 27839867
Evolutionary Homolog Study
The ability of cells to count and remember their divisions could underlie many alterations that occur during development, aging, and disease. This study tracked the cumulative divisional history of slow-cycling hematopoietic stem cells (HSCs) throughout adult life. A fraction of rarely dividing HSCs contained all the long-term HSC (LT-HSC) activity within the aging HSC compartment. During adult life, this population asynchronously completes four traceable symmetric self-renewal divisions to expand its size before entering a state of dormancy. The mechanism of expansion involves progressively lengthening periods between cell divisions, with long-term regenerative potential lost upon a fifth division. The data also show that age-related phenotypic changes within the HSC compartment are divisional history dependent. These results suggest that HSCs accumulate discrete memory stages over their divisional history and provide evidence for the role of cellular memory in HSC aging.
Signer, R. A., Qi, L., Zhao, Z., Thompson, D., Sigova, A. A., Fan, Z. P., DeMartino, G. N., Young, R. A., Sonenberg, N. and Morrison, S. J. (2016). The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.. Genes Dev 30: 1698-1703. PubMed ID: 27492367
Evolutionary Homolog Study
Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1; see Drosophila Thor) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis.

Thursday, December 15th

Fogle, K.J., Hertzler, J.I., Shon, J.H. and Palladino, M.J. (2016). The ATP-sensitive K channel is seizure protective and required for effective dietary therapy in a model of mitochondrial encephalomyopathy. J Neurogenet [Epub ahead of print]. PubMed ID: 27868454
Effective therapies are lacking for mitochondrial encephalomyopathies (MEs). MEs are devastating diseases that predominantly affect the energy-demanding tissues of the nervous system and muscle, causing symptoms such as seizures, cardiomyopathy, and neuro- and muscular degeneration. Even common anti-epileptic drugs which are frequently successful in ameliorating seizures in other diseases tend to have a lower success rate in ME, highlighting the need for novel drug targets, especially those that may couple metabolic sensitivity to neuronal excitability. Furthermore, alternative epilepsy therapies such as dietary modification are gaining in clinical popularity but have not been thoroughly studied in ME. Using the Drosophila ATP61 model of ME, this study analyzed dietary therapy throughout disease progression and found that it is highly effective against the seizures of ME, especially a high fat/ketogenic diet, and that the benefits are dependent upon a functional KATP channel complex. Further experiments with KATP show that it is seizure-protective in this model, and that pharmacological promotion of its open state also ameliorates seizures. These studies represent important steps forward in the development of novel therapies for a class of diseases that is notoriously difficult to treat, and lay the foundation for mechanistic studies of currently existing therapies in the context of metabolic disease.

Zhuang, N., Li, L., Chen, S. and Wang, T. (2016). PINK1-dependent phosphorylation of PINK1 and Parkin is essential for mitochondrial quality control. Cell Death Dis 7: e2501. PubMed ID: 27906179
Mitochondrial dysfunction has been linked to the pathogenesis of a large number of inherited diseases in humans, including Parkinson's disease, the second most common neurodegenerative disorder. The Parkinson's disease genes pink1 and parkin, which encode a mitochondrially targeted protein kinase, and an E3 ubiquitin ligase, respectively, participate in a key mitochondrial quality-control pathway that eliminates damaged mitochondria. This study established an in vivo PINK1/Parkin-induced photoreceptor neuron degeneration model in Drosophila with the aim of dissecting the PINK1/Parkin pathway in detail. Using LC-MS/MS analysis, Serine 346 was identified as the sole autophosphorylation site of Drosophila PINK1 and it was found that substitution of Serine 346 to Alanine completely abolishes the PINK1 autophosphorylation. Disruption of either PINK1 or Parkin phosphorylation impairs the PINK1/Parkin pathway, and the degeneration phenotype of photoreceptor neurons is obviously alleviated. Phosphorylation of PINK1 is not only required for the PINK1-mediated mitochondrial recruitment of Parkin but also induces its kinase activity toward Parkin. In contrast, phosphorylation of Parkin by PINK1 is dispensable for its translocation but required for its activation. Moreover, substitution with autophosphorylation-deficient PINK1 fails to rescue pink1 null mutant phenotypes. Taken together, these findings suggest that autophosphorylation of PINK1 is essential for the mitochondrial translocation of Parkin and for subsequent phosphorylation and activation of Parkin.

M'Angale, P. G. and Staveley, B. E. (2016). The HtrA2 Drosophila model of Parkinson's disease is suppressed by the pro-survival Bcl-2 Buffy. Genome: 1-7. PubMed ID: 27848260
Mutations in High temperature requirement A2 (HtrA2), also designated PARK13, which lead to the loss of its protease activity, have been associated with Parkinson's disease (PD). HtrA2 is a mitochondrial protease that translocates to the cytosol upon the initiation of apoptosis where it participates in the abrogation of inhibitors of apoptosis (IAP) inhibition of caspases. This study has demonstrated that the loss of the HtrA2 function in the dopaminergic neurons of Drosophila melanogaster results in PD-like phenotypes, and attempts were made to restore the age-dependent loss in locomotor ability by co-expressing the sole pro-survival Bcl-2 homologue Buffy. The inhibition of HtrA2 in the dopaminergic neurons of Drosophila resulted in shortened lifespan and impaired climbing ability, and the overexpression of Buffy rescued the reduction in lifespan and the age-dependent loss of locomotor ability. In supportive experiments, the inhibition of HtrA2 in the Drosophila eye results in eye defects, marked by reduction in ommatidia number and increased disruption of the ommatidial array; phenotypes that are suppressed by the overexpression of Buffy.
Bangi, E., Murgia, C., Teague, A.G., Sansom, O.J. and Cagan, R.L. (2016). Functional exploration of colorectal cancer genomes using Drosophila. Nat Commun 7: 13615. PubMed ID: 27897178
The multigenic nature of human tumours presents a fundamental challenge for cancer drug discovery. This study used Drosophila to generate 32 multigenic models of colon cancer using patient data from The Cancer Genome Atlas. These models recapitulate key features of human cancer, often as emergent properties of multigenic combinations. Multigenic models such as ras p53 pten apc exhibit emergent resistance to a panel of cancer-relevant drugs. Exploring one drug in detail, a mechanism of resistance for the PI3K pathway inhibitor BEZ235 was identified. Based on this, a combinatorial therapy that circumvents this resistance through a two-step process of emergent pathway dependence and sensitivity termed 'induced dependence' was developed. This approach is effective in cultured human tumour cells, xenografts and mouse models of colorectal cancer. These data demonstrate how multigenic animal models that reference cancer genomes can provide an effective approach for developing novel targeted therapies.

Wednesday, December 14th

Cavolo, S. L., Bulgari, D., Deitcher, D. L. and Levitan, E. S. (2016). Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles. J Neurosci 36(46): 11781-11787. PubMed ID: 27852784
Synaptic neuropeptide and neurotrophin stores are maintained by constitutive bidirectional capture of dense-core vesicles (DCVs) as they circulate in and out of the nerve terminal. Activity increases DCV capture to rapidly replenish synaptic neuropeptide stores following release. However, it is not known whether this is due to enhanced bidirectional capture. Experiments at the Drosophila neuromuscular junction, where DCVs contain neuropeptides and a bone morphogenic protein, show that activity-dependent replenishment of synaptic neuropeptides following release is evident after inhibiting the retrograde transport with the dynactin disruptor mycalolide B or photobleaching DCVs entering a synaptic bouton by retrograde transport. In contrast, photobleaching anterograde transport vesicles entering a bouton inhibits neuropeptide replenishment after activity. Furthermore, tracking of individual DCVs moving through boutons shows that activity selectively increases capture of DCVs undergoing anterograde transport. Finally, upregulating fragile X mental retardation 1 protein (Fmr1, also called FMRP) acts independently of futsch/MAP-1B to abolish activity-dependent, but not constitutive, capture. Fmr1 also reduces presynaptic neuropeptide stores without affecting activity-independent delivery and evoked release. Therefore, presynaptic motoneuron neuropeptide storage is increased by a vesicle capture mechanism that is distinguished from constitutive bidirectional capture by activity dependence, anterograde selectivity, and Fmr1 sensitivity. These results show that activity recruits a separate mechanism than used at rest to stimulate additional synaptic capture of DCVs for future release of neuropeptides and neurotrophins.
Vasin, A., Volfson, D., Littleton, J. T. and Bykhovskaia, M. (2016). Interaction of the Complexin accessory helix with Synaptobrevin regulates spontaneous fusion. Biophys J 111: 1954-1964. PubMed ID: 27806277
Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the plasma membrane. Vesicles attach to membranes via a specialized protein machinery composed of membrane-attached (t-SNARE; Syntaxin 1A) and vesicle-attached (v-SNARE; n-synaptobrevin) proteins that zipper together to form a coiled-coil SNARE bundle that brings the two fusing membranes into close proximity. Neurotransmitter release may occur either in response to an action potential or through spontaneous fusion. A cytosolic protein, Complexin (Cpx), binds the SNARE complex and restricts spontaneous exocytosis by acting as a fusion clamp. A model has been proposed in which the interaction between Cpx and the v-SNARE serves as a spring to prevent premature zippering of the SNARE complex, thereby reducing the likelihood of fusion. To test this model, molecular-dynamics (MD) simulations and site-directed mutagenesis of Cpx and SNAREs were combined in Drosophila. MD simulations of the Drosophila Cpx-SNARE complex demonstrated that Cpx's interaction with the v-SNARE promotes unraveling of the v-SNARE off the core SNARE bundle. Clamping properties were investigated in the syx3-69 paralytic mutant, which has a single-point mutation in the t-SNARE and displays enhanced spontaneous release. MD simulations demonstrated an altered interaction of Cpx with the SNARE bundle that hindered v-SNARE unraveling by Cpx, thus compromising clamping. This mode was used to predict mutations that should enhance the ability of Cpx to prevent full assembly of the SNARE complex. Transgenic Drosophila were generated with mutations in Cpx and the v-SNARE that disrupted a salt bridge between these two proteins. As predicted, both lines demonstrated a selective inhibition in spontaneous release, suggesting that Cpx acts as a fusion clamp that restricts full SNARE zippering.
Fontenas, L., De Santis, F., Di Donato, V., Degerny, C., Chambraud, B., Del Bene, F. and Tawk, M. (2016). Neuronal Ndrg4 is essential for nodes of Ranvier organization in zebrafish. PLoS Genet 12: e1006459. PubMed ID: 27902705
Evolutionary Homolog Study:
Axon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. This study identified N-myc downstream-regulated gene 4, ndrg4 (see Drosophila MESK2), as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 (see Drosophila Synapse protein 25) and nsf (see Drosophila Nsf2) are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery (see Drosophila Syntaxin 1A).
Lee, G. and Schwarz, T.L. (2016). Filamin, a synaptic organizer in Drosophila, determines glutamate receptor composition and membrane growth. Elife [Epub ahead of print]. PubMed ID: 27914199
Filamin is a scaffolding protein that functions in many cells as an actin-crosslinker. FLN90, an isoform of the Drosophila ortholog Filamin/cheerio that lacks the actin-binding domain, is shown in this study to govern the growth of postsynaptic membrane folds and the composition of glutamate receptor clusters at the larval neuromuscular junction. Genetic and biochemical analyses reveal that FLN90 is present surrounding synaptic boutons. FLN90 is required in the muscle for localization of the kinase dPak and, downstream of dPak, for localization of the GTPase Ral and the exocyst complex to this region. Consequently, Filamin is needed for growth of the subsynaptic reticulum. In addition, in the absence of filamin, type-A glutamate receptor subunits are lacking at the postsynapse, while type-B subunits cluster correctly. Receptor composition is dependent on dPak, but independent of the Ral pathway. Thus two major aspects of synapse formation, morphological plasticity and subtype-specific receptor clustering, require postsynaptic Filamin.

Tuesday, December 13th

Sanfilippo, P., Smibert, P., Duan, H. and Lai, E. C. (2016). Neural specificity of the RNA binding protein Elav is achieved by post-transcriptional repression in non-neural tissues. Development [Epub ahead of print]. PubMed ID: 27802174
Drosophila Elav is the founding member of the conserved family of Hu RNA binding proteins (RBPs), which collectively play critical and diverse roles in post-transcriptional regulation. Surprisingly, although Elav has a well-characterized neural cis-regulatory module, endogenous Elav is also ubiquitously transcribed and post-transcriptionally repressed in non-neural settings. In particular, mutant clones of multiple miRNA pathway components derepress ubiquitous Elav protein. Re-annotation of the elav transcription unit shows that not only does it generate extended 3' UTR isoforms, its universal 3' UTR isoform is much longer than previously believed. This longer common 3' UTR region includes multiple conserved, high-affinity sites for the miR-279/996 family. Notably, out of several miRNA mutants tested, endogenous Elav and a transgenic elav 3' UTR sensor are derepressed in mutant clones of mir-279/996, Cross-repression of Elav by another RBP was observed to be derepressed in non-neural miRNA pathway clones, namely Mei-P26. Finally, it was demonstrated that ubiquitous Elav has regulatory capacity, since derepressed Elav can stabilize an Elav-responsive sensor. It is critical to restrict Elav outside of the nervous system as misexpression of Elav in non-neural territories has profoundly adverse consequences. Altogether, this study defined unexpected post-transcriptional mechanisms that direct appropriate cell-type specific expression of a conserved neural RBP.
Xiong, X.P., Kurthkoti, K., Chang, K.Y., Li, J.L., Ren, X., Ni, J.Q., Rana, T.M. and Zhou, R. (2016). miR-34 modulates innate immunity and ecdysone signaling in Drosophila. PLoS Pathog 12: e1006034. PubMed ID: 27893816
microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. This study shows that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, there exists a mutual repression between miR-34 expression and ecdysone signaling, and miR-34 acts as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, the cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34 were identified. Taken together, these data enrich the repertoire of immune-modulating miRNAs in animals, and provide new insights into the interplay between steroid hormone signaling and innate immunity.

Vazquez-Pianzola, P., Schaller, B., Colombo, M., Beuchle, D., Neuenschwander, S., Marcil, A., Bruggmann, R. and Suter, B. (2016). The mRNA transportome of the Bicaudal D/Egalitarian transport machinery. RNA Biol [Epub ahead of print]. PubMed ID: 27801632
Messenger RNA (mRNA) transport focuses the expression of encoded proteins to specific regions within cells providing them with the means to assume specific functions and even identities. Bicaudal D and the mRNA binding protein Egalitarian interact with the microtubule motor dynein to localize mRNAs in Drosophila. Because relatively few mRNA cargos were known, Egl::GFP associated mRNAs were isolated and identified. The top candidates were validated by qPCR, in situ hybridization and genetically by showing that their localization requires BicD. In young embryos these Egl target mRNAs are preferentially localized apically, between the plasma membrane and the blastoderm nuclei, but also in the pole plasm at the posterior pole. Egl targets expressed in the ovary were mostly enriched in the oocyte and some were apically localized in follicle cells. The identification of a large group of novel mRNAs associated with BicD/Egl points to several novel developmental and physiological functions of this dynein dependent localization machinery. The verified dataset also allowed for development of a tool that predicts conserved A'-form-like stem loops that serve as localization elements in 3'UTRs.
Choudhury, S.R., Singh, A.K., McLeod, T., Blanchette, M., Jang, B., Badenhorst, P., Kanhere, A. and Brogna, S. (2016). Exon Junction Complex proteins bind nascent transcripts independently of pre-mRNA splicing in Drosophila melanogaster. Elife [Epub ahead of print]. PubMed ID: 27879206
Although it is currently understood that the exon junction complex (EJC) is recruited on spliced mRNA by a specific interaction between its central protein, eIF4AIII, and splicing factor CWC22, this study found that eIF4AIII and the other EJC core proteins Y14 and MAGO bind the nascent transcripts of not only intron-containing but also intronless genes on Drosophila polytene chromosome. Additionally, Y14 ChIP-seq demonstrates that association with transcribed genes is also splicing-independent in Drosophila S2 cells. The association of the EJC proteins with nascent transcripts does not require CWC22 and that of Y14 and MAGO is independent of eIF4AIII. eIF4AIII associates with both polysomal and monosomal RNA in S2 cell extracts, while Y14 and MAGO fractionate separately. Cumulatively, these data indicate a global role of eIF4AIII in gene expression, which would be independent of Y14 and MAGO, splicing, and of the EJC, as currently understood.

Monday, December 12th

Nemetschke, L. and Knust, E. (2016). Drosophila Crumbs prevents ectopic Notch activation in developing wings by inhibiting ligand-independent endocytosis. Development 143(23): 4543-4553. PubMed ID: 27899511
Many signalling components are apically restricted in epithelial cells, and receptor localisation and abundance is key for morphogenesis and tissue homeostasis. Hence, controlling apicobasal epithelial polarity is crucial for proper signalling. Notch is a ubiquitously expressed, apically localised receptor, which performs a plethora of functions; therefore, its activity has to be tightly regulated. This study shows that Drosophila Crumbs, an evolutionarily conserved polarity determinant, prevents Notch endocytosis in developing wings through direct interaction between the two proteins. Notch endocytosis in the absence of Crumbs results in the activation of the ligand-independent, Deltex-dependent Notch signalling pathway, and does not require the ligands Delta and Serrate or γ-secretase activity. This function of Crumbs is not due to general defects in apicobasal polarity, as localisation of other apical proteins is unaffected. These data reveal a mechanism to explain how Crumbs directly controls localisation and trafficking of the potent Notch receptor, and adds yet another aspect of Crumbs regulation in Notch pathway activity. Furthermore, the data highlight a close link between the apical determinant Crumbs, receptor trafficking and tissue homeostasis.
Thakur, R., Panda, A., Coessens, E., Raj, N., Yadav, S., Balakrishnan, S., Zhang, Q., Georgiev, P., Basak, B., Pasricha, R., Wakelam, M. J., Ktistakis, N. T. and Padinjat, R. (2016). Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling. Elife 5. PubMed ID: 27848911
During illumination, the light sensitive plasma membrane (rhabdomere) of Drosophila photoreceptors undergoes turnover with consequent changes in size and composition. However the mechanism by which illumination is coupled to rhabdomere turnover remains unclear. This study found that photoreceptors contain a light-dependent phospholipase D (PLD) activity. During illumination, loss of PLD resulted in an enhanced reduction in rhabdomere size, accumulation of Rab7 positive, rhodopsin1-containing vesicles (RLVs) in the cell body and reduced rhodopsin protein. These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD. In wild type photoreceptors, during illumination, enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function. Thus, during illumination, PLD activity couples endocytosis of RLVs with their recycling to the plasma membrane thus maintaining plasma membrane size and composition.
Woods, J. K., Ziafazeli, T. and Rogina, B. (2016). Rpd3 interacts with insulin signaling in Drosophila longevity extension. Aging (Albany NY) [Epub ahead of print]. PubMed ID: 27852975
Histone deacetylase (HDAC) 1 regulates chromatin compaction and gene expression by removing acetyl groups from lysine residues within histones. HDAC1 affects a variety of processes including proliferation, development, metabolism, and cancer. Reduction or inhibition of Rpd3, yeast and fly HDAC1 orthologue, extends longevity. However, the mechanism of rpd3's effects on longevity remains unclear. This study reports an overlap between rpd3 and the Insulin/Insulin-like growth factor signaling (IIS) longevity pathways. rpd3 reduction downregulates expression of members of the IIS pathway, which is associated with altered metabolism, increased energy storage, and higher resistance to starvation and oxidative stress. Genetic studies support the role of IIS in rpd3 longevity pathway, as illustrated with reduced stress resistance and longevity of flies double mutant for rpd3 and dfoxo, a downstream target of IIS pathway, compared to rpd3 single mutant flies. The data suggest that increased dfoxo is a mediator of rpd3 's effects on fly longevity and intermediary metabolism, and confer a new link between rpd3 and IIS longevity pathways.
Lüdtke TH, et al. (2016). Tbx2 and Tbx3 act downstream of Shh to maintain canonical Wnt signaling during branching morphogenesis of the murine lung. Dev Cell 39(2): 239-253. PubMed ID: 27720610
Evolutionary Homolog Study
Numerous signals drive the proliferative expansion of the distal endoderm and the underlying mesenchyme during lung branching morphogenesis, but little is known about how these signals are integrated. This study demonstrates by analysis of conditional double mutants that the two T-box transcription factor genes Tbx2 and Tbx3 (see Drosophila Bifid/Optomotor blind) act together in the lung mesenchyme to maintain branching morphogenesis. Expression of both genes depends on epithelially derived Shh signaling (see Drosophila Hedgehog), with additional modulation by Bmp, Wnt, and Tgfβ signaling. Genetic rescue experiments reveal that Tbx2 and Tbx3 function downstream of Shh to maintain pro-proliferative mesenchymal Wnt signaling, in part by direct repression of the Wnt antagonists Frzb and Shisa3. In combination with a previous finding that Tbx2 and Tbx3 repress the cell-cycle inhibitors Cdkn1a and Cdkn1b, it is concluded that Tbx2 and Tbx3 maintain proliferation of the lung mesenchyme by way of at least two molecular mechanisms: regulating cell-cycle regulation and integrating the activity of multiple signaling pathways.

Sunday, December 11th

Li, J., Terry, E.E., Fejer, E., Gamba, D., Hartmann, N., Logsdon, J., Michalski, D., Rois, L.E., Scuderi, M.J., Kunst, M. and Hughes, M.E. (2016). Achilles is a circadian clock-controlled gene that regulates immune function in Drosophila. Brain Behav Immun [Epub ahead of print]. PubMed ID: 27856350
The circadian clock is a transcriptional/translational feedback loop that drives the rhythmic expression of downstream mRNAs. Termed "clock-controlled genes," these molecular outputs of the circadian clock orchestrate cellular, metabolic, and behavioral rhythms. This study identified a novel clock-controlled gene in Drosophila melanogaster, Achilles (Achl), which is rhythmic at the mRNA level in the brain and which represses expression of anti-microbial peptides in the immune system. Achilles knock-down in neurons dramatically elevates expression of crucial immune response genes, including IM1 (Immune induced molecule 1), Mtk (Metchnikowin), and Drs (Drosomysin). As a result, flies with knocked-down Achilles expression are resistant to bacterial challenges. Meanwhile, no significant change in core clock gene expression and locomotor activity is observed, suggesting that Achilles influences rhythmic mRNA outputs rather than directly regulating the core timekeeping mechanism. Notably, Achilles knock-down in the absence of immune challenge significantly diminishes the fly's overall lifespan, indicating a behavioral or metabolic cost of constitutively activating this pathway. Together, these data demonstrate that (1) Achilles is a novel clock-controlled gene that (2) regulates the immune system, and (3) participates in signaling from neurons to immunological tissues.

Iatsenko, I., Kondo, S., Mengin-Lecreulx, D. and Lemaitre, B. (2016). PGRP-SD, an Extracellular Pattern-Recognition Receptor, enhances peptidoglycan-mediated activation of the Drosophila Imd pathway. Immunity 45: 1013-1023. PubMed ID: 27851910
Activation of the innate immune response in metazoans is initiated through the recognition of microbes by host pattern-recognition receptors. In Drosophila, diaminopimelic acid (DAP)-containing peptidoglycan from Gram-negative bacteria is detected by the transmembrane receptor Peptidoglycan recognition protein LC (PGRP-LC) and by the intracellular receptor PGRP-LE. This study shows that PGRP-SD acted upstream of PGRP-LC as an extracellular receptor to enhance peptidoglycan-mediated activation of Imd signaling. Consistent with this, PGRP-SD mutants exhibited impaired activation of the Imd pathway and increased susceptibility to DAP-type bacteria. PGRP-SD enhanced the localization of peptidoglycans to the cell surface and hence promoted signaling. Moreover, PGRP-SD antagonized the action of PGRP-LB, an extracellular negative regulator, to fine-tune the intensity of the immune response. These data reveal that Drosophila PGRP-SD functions as an extracellular receptor similar to mammalian CD14 and demonstrate that, comparable to lipopolysaccharide sensing in mammals, Drosophila relies on both intra- and extracellular receptors for the detection of bacteria.
Early, A. M., Arguello, J. R., Cardoso-Moreira, M., Gottipati, S., Grenier, J. K. and Clark, A. G. (2016). Survey of global genetic diversity within the Drosophila immune system. Genetics [Epub ahead of print]. PubMed ID: 27815361
Numerous studies across a wide range of taxa have demonstrated that immune genes are routinely among the most rapidly evolving genes in the genome. This observation, however, does not address what proportion of immune genes undergo strong selection during adaptation to novel environments. This study determined the extent of very recent divergence in genes with immune function across five populations of Drosophila melanogaster;t immune genes do not show an overall trend of recent rapid adaptation. The population-based approach uses a set of carefully matched control genes to account for the effects of demography and local recombination rate, allowing identification of whether specific immune functions are putative targets of strong selection. Evidence was found that viral defense genes are rapidly evolving in Drosophila at multiple time scales. Local adaptation to bacteria and fungi is less extreme and primarily occurs through changes in recognition and effector genes rather than large-scale changes to the regulation of the immune response. Surprisingly, genes in the Toll pathway, which show a high rate of adaptive substitutions between the D. melanogaster and D. simulans lineages, show little population differentiation. Quantifying the flies for resistance to a generalist Gram-positive bacterial pathogen, it was found that this genetic pattern of low population differentiation was recapitulated at the phenotypic level. In sum, these results highlight the complexity of immune evolution and suggest that Drosophila immune genes do not follow a uniform trajectory of strong directional selection as flies encounter new environments.
Ye, Y.H., Seleznev, A., Flores, H.A., Woolfit, M. and McGraw, E.A. (2017). Gut microbiota in Drosophila melanogaster interacts with Wolbachia but does not contribute to Wolbachia-mediated antiviral protection. J Invertebr Pathol [Epub ahead of print]. PubMed ID: 27871813
Animals experience near constant infection with microorganisms. A significant proportion of these microbiota reside in the alimentary tract. There is a growing appreciation for the roles gut microbiota play in host biology. The gut microbiota of insects, for example, have been shown to help the host overcome pathogen infection either through direct competition or indirectly by stimulating host immunity. These defenses may also be supplemented by coinfecting maternally inherited microbes such as Wolbachia. The presence of Wolbachia in a host can delay and/or reduce death caused by RNA viruses. Whether the gut microbiota of the host interacts with Wolbachia, or vice versa, the precise role of Wolbachia in antiviral protection is not known. This study used 16S rDNA sequencing to characterize changes in gut microbiota composition in Drosophila melanogaster associated with Wolbachia infection and antibiotic treatment. Subsequently, it was tested whether changes in gut composition via antibiotic treatment alter Wolbachia-mediated antiviral properties. It was found that both antibiotics and Wolbachia significantly reduce the biodiversity of the gut microbiota without changing the total microbial load. Changing the gut microbiota composition with antibiotic treatment enhances Wolbachia density but does not confer greater antiviral protection against Drosophila C virus to the host. In conclusion, there are significant interactions between Wolbachia and gut microbiota, but changing gut microbiota composition is not likely to be a means through which Wolbachia conveys antiviral protection to its host.

Saturday, December 10th

Porcelli, D., Gaston, K.J., Butlin, R.K. and Snook, R.R. (2016). Local adaptation of reproductive performance during thermal stress. J Evol Biol [Epub ahead of print]. PubMed ID: 27862539
Considerable evidence exists for local adaptation of critical thermal limits in ectotherms following adult temperature stress, but fewer studies have tested for local adaptation of sublethal heat stress effects across life-history stages. In organisms with complex life cycles, such as holometabolous insects, heat stress during juvenile stages may severely impact gametogenesis, having downstream consequences on reproductive performance that may be mediated by local adaptation, although this is rarely studied. This study tested how exposure to either benign or heat stress temperature during juvenile and adult stages, either independently or combined, influences egg-to-adult viability, adult sperm motility and fertility in high- and low-latitude populations of Drosophila subobscura. Both population- and temperature-specific effects on survival and sperm motility were found- juvenile heat stress decreases survival and subsequent sperm motility and each trait is lower in the northern population. An interaction between population and temperature on fertility following application of juvenile heat stress was observed; although fertility is negatively impacted in both populations, the southern population is less affected. When the adult stage was subjected to heat stress, the southern population was found to exhibit positive carry-over effects whereas the northern population's fertility remained low. Thus, the northern population is more susceptible to sublethal reproductive consequences following exposure to juvenile heat stress. This may be common in other organisms with complex life cycles and current models predicting population responses to climate change, which do not take into account the impact of juvenile heat stress on reproductive performance, may be too conservative.

Vigoder, F. M., Parker, D. J., Cook, N., Tourniere, O., Sneddon, T. and Ritchie, M. G. (2016). Inducing Cold-Sensitivity in the Frigophilic Fly Drosophila montana by RNAi. PLoS One 11: e0165724. PubMed ID: 27832122
Cold acclimation is a critical physiological adaptation for coping with seasonal cold. By increasing their cold tolerance individuals can remain active for longer at the onset of winter and can recover more quickly from a cold shock. In insects, despite many physiological studies, little is known about the genetic basis of cold acclimation. Recently, transcriptomic analyses in Drosophila virilis and D. montana revealed candidate genes for cold acclimation by identifying genes upregulated during exposure to cold. This study tested the role of myo-inositol-1-phosphate synthase (Inos), in cold tolerance in D. montana using an RNAi approach. D. montana has a circumpolar distribution and overwinters as an adult in northern latitudes with extreme cold. Cold tolerance of dsRNA knock-down flies was tested using two metrics: chill-coma recovery time (CCRT) and mortality rate after cold acclimation. Injection of dsRNAInos did not alter CCRT, either overall or in interaction with the cold treatment, however it did induced cold-specific mortality, with high levels of mortality observed in injected flies acclimated at 5 degrees C but not at 19 degrees C. Overall, injection with dsRNAInos induced a temperature-sensitive mortality rate of over 60% in this normally cold-tolerant species. qPCR analysis confirmed that dsRNA injection successfully reduced gene expression of Inos. Thus, these results demonstrate the involvement of Inos in increasing cold tolerance in D. montana. The potential mechanisms involved by which Inos increases cold tolerance are also discussed.
Vozza, A., De Leonardis, F., Paradies, E., De Grassi, A., Pierri, C. L., Parisi, G., Marobbio, C. M., Lasorsa, F. M., Muto, L., Capobianco, L., Dolce, V., Raho, S. and Fiermonte, G. (2016). Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 27836698
CoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported in this study and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.
Nelson, C. S., Beck, J. N., Wilson, K. A., Pilcher, E. R., Kapahi, P. and Brem, R. B. (2016). Cross-phenotype association tests uncover genes mediating nutrient response in Drosophila. BMC Genomics 17: 867. PubMed ID: 27809764
Obesity-related diseases are major contributors to morbidity and mortality in the developed world. Molecular diagnostics and targets of therapies to combat nutritional imbalance are urgently needed in the clinic. Invertebrate animals have been a cornerstone of basic research efforts to dissect the genetics of metabolism and nutrient response. This study used fruit flies reared on restricted and nutrient-rich diets to identify genes associated with starvation resistance, body mass and composition, in a survey of genetic variation across the Drosophila Genetic Reference Panel (DGRP). Starvation resistance, body weight and composition were measured in DGRP lines on each of two diets, and several association mapping strategies were used to harness this panel of phenotypes for molecular insights. DNA sequence variants were tested for a relationship with single metabolic traits and with multiple traits at once, using a scheme for cross-phenotype association mapping; association tests focused on homologs of human disease genes and common polymorphisms; and gene-by-diet interactions were tested. The results revealed gene and gene-by-diet associations between 17 variants and body mass, whole-body triglyceride and glucose content, or starvation resistance. Focused molecular experiments validated the role in body mass of an uncharacterized gene, CG43921 (which was rename heavyweight), and previously unknown functions for the diacylglycerol kinase rdgA, the huntingtin homolog htt, and the ceramide synthase schlank in nutrient-dependent body mass, starvation resistance, and lifespan. The findings implicate a wealth of gene candidates in fly metabolism and nutrient response, and ascribe novel functions to htt, rdgA, hwt and schlank.

Friday, December 9th

Ryu, T., Bonner, M. and Chiolo, I. (2016). Cervantes and Quijote protect heterochromatin from aberrant recombination and lead the way to the nuclear periphery. Nucleus [Epub ahead of print] PubMed ID: 27673416
Repairing double-strand breaks (DSBs) is particularly challenging in heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination and chromosome rearrangements. In Drosophila cells, faithful homologous recombination (HR) repair of heterochromatic DSBs relies on a specialized pathway that relocalizes repair sites to the nuclear periphery before Rad51 recruitment. This study shows that HR progression is initially blocked inside the heterochromatin domain by SUMOylation and the coordinated activity of two distinct Nse2 SUMO E3 ligases: Quijote (Qjt) and Cervantes (Cerv). In addition, the SUMO-targeted ubiquitin ligase (STUbL) Dgrn, but not its partner dRad60, is recruited to heterochromatic DSBs at early stages of repair and mediates relocalization. However, Dgrn is not required to prevent Rad51 recruitment inside the heterochromatin domain, suggesting that the block to HR progression inside the domain and relocalization to the nuclear periphery are genetically separable pathways. Further, SUMOylation defects affect relocalization without blocking heterochromatin expansion, revealing that expansion is not required for relocalization. Finally, nuclear pores and inner nuclear membrane proteins (INMPs) anchor STUbL/RENi components and repair sites to the nuclear periphery, where repair continues. Together, these studies reveal a critical role of SUMOylation and nuclear architecture in the spatial and temporal regulation of heterochromatin repair and the protection of genome integrity.
Xia, B., Gerstin, E., Schones, D.E., Huang, W. and Steven de Belle, J. (2016). Transgenerational programming of longevity through E(z)-mediated histone H3K27 trimethylation in Drosophila. Aging (Albany NY) 8: 2988-3008. PubMed ID: 27889707
Transgenerational effects on health and development of early-life nutrition have gained increased attention recently. However, the underlying mechanisms of transgenerational transmission are only starting to emerge, with epigenetics as perhaps the most important mechanism. The first Drosophila model to study transgenerational programming of longevity after early-life dietary manipulations has been previously reported, enabling investigations to identify underlying epigenetic mechanisms. This study reports that post-eclosion dietary manipulation (PDM) with a low-protein (LP) diet upregulates the protein level of E(z), an H3K27 specific methyltransferase, leading to higher levels of H3K27 trimethylation (H3K27me3). This PDM-mediated change in H3K27me3 corresponds with a shortened longevity of F0 flies as well as their F2 offspring. Specific RNAi-mediated post-eclosion knockdown of E(z) or pharmacological inhibition of its enzymatic function with EPZ-6438 in the F0 parents improves longevity while rendering H3K27me3 low across generations. Importantly, addition of EPZ-6438 to the LP diet fully alleviates the longevity-reducing effect of the LP PDM, supporting the increased level of E(z)-dependent H3K27me3 as the primary cause and immediate early-life period as the critical time to program longevity through epigenetic regulation. These observations establish E(z)-mediated H3K27me3 as one epigenetic mechanism underlying nutritional programming of longevity and support the use of EPZ-6438 to extend lifespan.

Hitrik, A., Popliker, M., Gancz, D., Mukamel, Z., Lifshitz, A., Schwartzman, O., Tanay, A. and Gilboa, L. (2016). Combgap promotes ovarian niche development and chromatin association of EcR-binding regions in BR-C. PLoS Genet 12: e1006330. PubMed ID: 27846223
The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. This study shows that the nuclear protein Combgap (Cg) supports correct ovarian niche formation in Drosophila by controlling Ecdysone-Receptor (EcR)- mediated transcription and long-range chromatin contacts in the broad locus (BR-C). Both cg and BR-C promote ovarian growth and the development of niches for germ line stem cells. BR-C levels were lower when Combgap was either reduced or over-expressed, indicating an intricate regulation of the BR-C locus by Combgap. Polytene chromosome stains showed that Cg co-localizes with EcR, the major regulator of BR-C, at the BR-C locus and that EcR binding to chromatin was sensitive to changes in Cg levels. Proximity ligation assay indicated that the two proteins could reside in the same complex. Finally, chromatin conformation analysis revealed that EcR-bound regions within BR-C, which span ~30 KBs, contacted each other. Significantly, these contacts were stabilized in an ecdysone- and Combgap-dependent manner. Together, these results highlight Combgap as a novel regulator of chromatin structure that promotes transcription of ecdysone target genes and ovarian niche formation.
Roelens, B., Clemot, M., Leroux-Coyau, M., Klapholz, B. and Dostatni, N. (2016). Maintenance of heterochromatin by the large subunit of the replication-coupled histone chaperone CAF-1 requires its interaction with HP1a through a conserved motif. Genetics [Epub ahead of print]. PubMed ID: 27838630
In eukaryotic cells, the organization of genomic DNA into chromatin regulates many biological processes, from the control of gene expression to the regulation of chromosome segregation. The proper maintenance of this structure upon cell division is therefore of prime importance during development for the maintenance of cell identity and genome stability. The Chromatin Assembly Factor 1 (CAF-1) is involved in the assembly of H3-H4 histone dimers on newly synthesized DNA and in the maintenance of a higher order structure, the heterochromatin, through an interaction of its large subunit with the heterochromatin protein HP1a. This study identified a conserved domain in the large subunit of the CAF-1 complex required for its interaction with HP1a in the Drosophila fruit fly. Functional analysis reveals that this domain is dispensable for viability but participates in two processes involving heterochromatin: position-effect variegation (PEV) and long range chromosomal interactions during meiotic prophase. Importantly, the identification in the large subunit of CAF-1 of a domain required for its interaction with HP1 allows the separation of its functions in heterochromatin related processes from its function in the assembly of H3-H4 dimers onto newly synthesized DNA.

Thursday, December 8th

Saur, T., Peng, I.F., Jiang, P., Gong, N., Yao, W.D., Xu, T.L. and Wu, C.F. (2016). K+ channel reorganization and homeostatic plasticity during postembryonic development: biophysical and genetic analyses in acutely dissociated Drosophila central neurons. J Neurogenet [Epub ahead of print]. PubMed ID: 27868467
Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, this study shows drastic re-assemblies and compensatory regulations of voltage-gated (IKv) and Ca2+-activated (IK(Ca)) K+ currents during postembryonic development. Larval and adult neurons display prominent fast-inactivating IKv, mediated by the Shaker (Sh) channel to a large extent, while in the same neurons IK(Ca) is far smaller in amplitude. In contrast, pupal neurons are characterized by large sustained IKv and prominent IK(Ca), encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the ShM null mutant removes inactivating, transient IKv from large portions of neurons at all stages. Interestingly, elimination of Sh currents is accompanied by upregulation of non-Sh transient IKv. In comparison, the slo1 mutation abolishes the vast majority of IK(Ca), particularly at the pupal stage. Strikingly, the deficiency of IK(Ca) in slo pupae is compensated by the transient component of IKv mediated by Sh channels. Thus, IK(Ca) appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient IKv current. While mutants lacking either Sh or slo currents survive normally, Sh;;slo double mutants deficient in both fail to survive through pupal metamorphosis. Together, these data highlight significant reorganizations and homeostatic compensations of K+ currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process.

Lang, P. Y., Nanjangud, G. J., Sokolsky-Papkov, M., Shaw, C., Hwang, D., Parker, J. S., Kabanov, A. V. and Gershon, T. R. (2016). ATR maintains chromosomal integrity during postnatal cerebellar neurogenesis and is required for medulloblastoma formation. Development 143(21): 4038-4052. PubMed ID: 27803059
Evolutionary Homolog Study
Microcephaly and medulloblastoma may both result from mutations that compromise genomic stability. This study reports that ATR (see Drosophila ATR), which is mutated in the microcephalic disorder Seckel syndrome, sustains cerebellar growth by maintaining chromosomal integrity during postnatal neurogenesis. Atr deletion in cerebellar granule neuron progenitors (CGNPs) induced proliferation-associated DNA damage, p53 activation (see Drosophila p53), apoptosis and cerebellar hypoplasia in mice. Co-deletions of either p53 or Bax and Bak prevented apoptosis in Atr-deleted CGNPs, but failed to fully rescue cerebellar growth. ATR-deficient CGNPs had impaired cell cycle checkpoint function and continued to proliferate, accumulating chromosomal abnormalities. RNA-Seq demonstrated that the transcriptional response to ATR-deficient proliferation was highly p53 dependent and markedly attenuated by p53 co-deletion. Acute ATR inhibition in vivo by nanoparticle-formulated VE-822 reproduced the developmental disruptions seen with Atr deletion. Genetic deletion of Atr blocked tumorigenesis in medulloblastoma-prone SmoM2 mice. These data show that p53-driven apoptosis and cell cycle arrest - and, in the absence of p53, non-apoptotic cell death - redundantly limit growth in ATR-deficient progenitors. These mechanisms may be exploited for treatment of CGNP-derived medulloblastoma using ATR inhibition.
van Giesen, L., Neagu-Maier, G. L., Kwon, J. Y. and Sprecher, S. G. (2016). A microfluidics-based method for measuring neuronal activity in Drosophila chemosensory neurons. Nat Protoc 11: 2389-2400. PubMed ID: 27809317
Monitoring neuronal responses to defined sensory stimuli is a powerful and widely used approach for understanding sensory coding in the nervous system. However, providing precise, stereotypic and reproducible cues while concomitantly recording neuronal activity remains technically challenging. This study describes the fabrication and use of a microfluidics system that allows precise temporally restricted stimulation of Drosophila chemosensory neurons with an array of different chemical cues. The system can easily be combined with genetically encoded calcium sensors, and it can measure neuronal activity at single-cell resolution in larval sense organs and in the proboscis or leg of the adult fly. The design of the master mold is described, along with the production of the microfluidic chip and live imaging using the calcium sensor GCaMP, expressed in distinct types of Drosophila chemosensory neurons. Fabrication of the master mold and microfluidic chips requires basic skills in photolithography and takes approximately 2 weeks; the same devices can be used repeatedly over several months. Flies can be prepared for measurements in minutes and imaged for up to 1 h.
Gong, J., Yuan, Y., Ward, A., Kang, L., Zhang, B., Wu, Z., Peng, J., Feng, Z., Liu, J. and Xu, X. Z. (2016). The C. elegans taste receptor homolog LITE-1 is a photoreceptor. Cell 167(5): 1252-1263 e1210. PubMed ID: 27863243
Evolutionary Homolog Study
Many animal tissues/cells are photosensitive, yet only two types of photoreceptors (i.e., opsins and cryptochromes) have been discovered in metazoans. The question arises as to whether unknown types of photoreceptors exist in the animal kingdom. LITE-1, a seven-transmembrane gustatory receptor (GR) homolog, mediates UV-light-induced avoidance behavior in C. elegans. However, it is not known whether LITE-1 functions as a chemoreceptor or photoreceptor. This study shows that LITE-1 directly absorbs both UVA and UVB light with an extinction coefficient 10-100 times that of opsins and cryptochromes, indicating that LITE-1 is highly efficient in capturing photons. Unlike typical photoreceptors employing a prosthetic chromophore to capture photons, LITE-1 strictly depends on its protein conformation for photon absorption. Two tryptophan residues critical for LITE-1 function were identified. Interestingly, unlike GPCRs, LITE-1 adopts a reversed membrane topology. Thus, LITE-1, a taste receptor homolog, represents a distinct type of photoreceptor in the animal kingdom.

Wednesday, December 7th

Walck-Shannon, E., Lucas, B., Chin-Sang, I., Reiner, D., Kumfer, K., Cochran, H., Bothfeld, W. and Hardin, J. (2016). CDC-42 orients cell migration during epithelial intercalation in the Caenorhabditis elegans epidermis. PLoS Genet 12: e1006415. PubMed ID: 27861585
Evolutionary Homolog Study:
Cell intercalation is a highly directed cell rearrangement that is essential for animal morphogenesis. As such, intercalation requires orchestration of cell polarity across the plane of the tissue. CDC-42 is a Rho family GTPase with key functions in cell polarity, yet its role during epithelial intercalation has not been established because its roles early in embryogenesis have historically made it difficult to study. To circumvent these early requirements, this study used tissue-specific and conditional loss-of-function approaches to identify a role for CDC-42 (see Drosophila Cdc42) during intercalation of the Caenorhabditis elegans dorsal embryonic epidermis (see Drosophila morphogenetic movements). CDC-42 activity is enriched in the medial tips of intercalating cells, which extend as cells migrate past one another. Moreover, CDC-42 is involved in both the efficient formation and orientation of cell tips during cell rearrangement. Using conditional loss-of-function it was shown that the PAR complex (see Drosophila PAR complex) functions in tip formation and orientation. Additionally, the sole C. elegans Eph receptor, VAB-1 (see Drosophila Eph), was found to function during this process in an Ephrin-independent manner. Using epistasis analysis, it was shown that vab-1 lies in the same genetic pathway as cdc-42 and is responsible for polarizing CDC-42 activity to the medial tip. Together, these data establish a previously uncharacterized role for polarized CDC-42, in conjunction with PAR-6, PAR-3 and an Eph receptor, during epithelial intercalation.

Ikeda, T. and Satou, Y. (2016). Differential temporal control of Foxa.a and Zic-r.b specifies brain versus notochord fate in the ascidian embryo. Development [Epub ahead of print]. PubMed ID: 27888196
Evolutionary Homolog Study
In embryos of an invertebrate chordate, Ciona intestinalis, two transcription factors, Foxa.a (see Drosophila Foxa) and Zic-r.b, (see Drosophila Odd-paired) are required for specification of the brain and the notochord, which are derived from distinct cell lineages. In the brain lineage, Foxa.a and Zic-r.b are expressed with no temporal overlap. In the notochord lineage, Foxa.a and Zic-r.b are expressed simultaneously. In the present study found that the temporally non-overlapping expression of Foxa.a and Zic-r.b in the brain lineage was regulated by three repressors, Prdm1-r.a and Prdm1-r.b ) (see Drosophila Hamlet) and Hes.a (see Drosophila Hairy). In morphant embryos of these three repressor genes, Foxa.a expression was not terminated at the normal time, in addition to precocious expression of Zic-r.b Consequently, Foxa.a and Zic-r.b were expressed simultaneously, which led to ectopic activation of Brachyury (see Drosophila Brachyury) and its downstream pathways for notochord differentiation. Thus, temporal controls by transcriptional repressors are essential for specifying the two distinct fates of brain and notochord by Foxa.a and Zic-r.b. Such a mechanism might enable the repeated use of a limited repertoire of transcription factors in developmental gene regulatory networks.
Duque, J. and Gorfinkiel, N. (2016). Integration of actomyosin contractility with cell-cell adhesion during dorsal closure. Development [Epub ahead of print]. PubMed ID: 27836966
This work investigated the morphogenesis of the amnioserosa, the main force-generating tissue during the process of dorsal closure in Drosophila. Myosin activity was shown to determine the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. In contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, in AS cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through the control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis.

Navarrete, I. A. and Levine, M. (2016). Nodal and FGF coordinate ascidian neural tube morphogenesis. Development [Epub ahead of print]. PubMed ID: 27827820
Evolutionary Homolog Study
This study investigated the formation of the neural tube in Ciona intestinalis. Previous studies have implicated Nodal and FGF signals in the specification of lateral and ventral neural progenitors. This study showed that these signals also control the detailed cellular behaviors underlying morphogenesis of the neural tube. Live imaging experiments show that FGF (see Drosophila Branchless) controls the intercalary movements of ventral neural progenitors, while Nodal is essential for the characteristic stacking behavior of lateral cells. Ectopic activation of FGF signaling is sufficient to induce intercalary behaviors in cells that have not received Nodal. In the absence of FGF and Nodal, neural progenitors exhibit a default behavior of sequential cell divisions, and fail to undergo the intercalary and stacking behaviors essential for normal morphogenesis. Thus, cell specification events occurring prior to completion of gastrulation coordinate morphogenetic movements underlying the organization of the neural tube.
Sacilotto, N., Chouliaras, K.M., Nikitenko, L.L., Lu, Y.W., Fritzsche, M., Wallace, M.D., Nornes, S., García-Moreno, F., Payne, S., Bridges, E., Liu, K., Biggs, D., Ratnayaka, I., Herbert, S.P., Moln├ír, Z., Harris, A.L., Davies, B., Bond, G.L., Bou-Gharios, G., Schwarz, J.J. and De Val, S. (2016). MEF2 transcription factors are key regulators of sprouting angiogenesis. Genes Dev 30: 2297-2309. PubMed ID: 27898394
Evolutionary Homolog Study
Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis (see Drosophila dorsal vessel), the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) (see Drosophila Pvf1) and Delta-like ligand 4 (Dll4)/Notch signaling (see Drosophila Notch) and requires high levels of Notch ligand DLL4 (see Drosophila Dl). This study identifies MEF2 (see Drosophila Mef2) transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, it was found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 (see Drosophila nej) to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.

Tuesday, December 6th

Srinivasan, N., Gordon, O., Ahrens, S., Franz, A., Deddouche, S., Chakravarty, P., Phillips, D., Yunus, A.A., Rosen, M.K., Valente, R.S., Teixeira, L., Thompson, B., Dionne, M.S., Wood, W., Reis, E. and Sousa, C. (2016). Actin is an evolutionarily-conserved damage-associated molecular pattern that signals tissue injury in Drosophila melanogaster. Elife [Epub ahead of print]. PubMed ID: 27871362
Damage associated molecular patterns (DAMPs) are released by dead cells and can trigger sterile inflammation and, in vertebrates, adaptive immunity. Actin is a DAMP detected in mammals by the receptor, DNGR-1, expressed by dendritic cells (DCs). DNGR-1 is phosphorylated by Src-family kinases and recruits the tyrosine kinase Syk to promote DC cross-presentation of dead cell-associated antigens. This study reports that actin is also a DAMP in invertebrates that lack DCs and adaptive immunity. Administration of actin to Drosophila melanogaster triggers a response characterised by selective induction of STAT target genes in the fat body through the cytokine Upd3 and its JAK/STAT-coupled receptor, Domeless. Notably, this response requires signalling via Shark, the Drosophila orthologue of Syk, and Src42A, a Drosophila Src-family kinase, and is dependent on Nox activity. Thus, extracellular actin detection via a Src-family kinase-dependent cascade is an ancient means of detecting cell injury that precedes evolution of adaptive immunity.

Lin, Z., Guo, H., Cao, Y., Zohrabian, S., Zhou, P., Ma, Q., VanDusen, N., Guo, Y., Zhang, J., Stevens, S.M., Liang, F., Quan, Q., van Gorp, P.R., Li, A., Dos Remedios, C., He, A., Bezzerides, V.J. and Pu, W.T. (2016). Acetylation of VGLL4 regulates Hippo-YAP signaling and postnatal cardiac growth. Dev Cell 39: 466-479. PubMed ID: 27720608
Evolutionary Homolog Study:
Binding of the transcriptional co-activator YAP (see Drosophila yki) with the transcription factor TEAD (see Drosophila sd) stimulates growth of the heart (see Drosophila cardiac development) and other organs. YAP overexpression potently stimulates fetal cardiomyocyte (CM) proliferation, but YAP's mitogenic potency declines postnatally. While investigating factors that limit YAP's postnatal mitogenic activity, this study found that the CM-enriched TEAD1 binding protein VGLL4 (see Drosophila CG1737) inhibits CM proliferation by inhibiting TEAD1-YAP interaction and by targeting TEAD1 for degradation. Importantly, VGLL4 acetylation at lysine 225 negatively regulates its binding to TEAD1. This developmentally regulated acetylation event critically governs postnatal heart growth, since overexpression of an acetylation-refractory VGLL4 mutant enhances TEAD1 degradation, limits neonatal CM proliferation, and causes CM necrosis. These observations define an acetylation-mediated, VGLL4-dependent switch that regulates TEAD stability and YAP-TEAD activity. These insights may improve targeted modulation of TEAD-YAP activity in applications from cardiac regeneration to cancer.
Gargini R, Escoll M, García E, García-Escudero R, Wandosell F, Antón IM. (2016). WIP drives tumor progression through YAP/TAZ-dependent autonomous cell growth. Cell Rep. 17(8):1962-1977. PubMed ID: 27851961
Evolutionary Homolog Study
In cancer, the deregulation of growth signaling pathways drives changes in the cell's architecture and its environment that allow autonomous growth of tumors. These cells then acquire a tumor-initiating "stemness" phenotype responsible for disease advancement to more aggressive stages. This study shows that high levels of the actin cytoskeleton-associated protein WIP (WASP-interacting protein; see Drosophila Verprolin) correlates with tumor growth, both of which are linked to the tumor-initiating cell phenotype. WIP controls tumor growth by boosting signals that stabilize the YAP/TAZ complex (see Drosophila Yorkie) via a mechanism mediated by the endocytic/endosomal system. When WIP levels are high, the β-catenin Adenomatous polyposis coli (APC)-axin-GSK3 destruction complex (see Drosophila Apc) is sequestered to the multi-vesicular body compartment, where its capacity to degrade YAP/TAZ is inhibited. YAP/TAZ stability is dependent on Rac (see Drosophila Rac1), p21-activated kinase (PAK) and mammalian diaphanous-related formin (mDia; see Drosophila Diaphanous), and is Hippo independent. This close biochemical relationship indicates an oncogenic role for WIP in the physiology of cancer pathology by increasing YAP/TAZ stability.
Lüdtke, T.H., Rudat, C., Wojahn, I., Weiss, A.C., Kleppa, M.J., Kurz, J., Farin, H.F., Moon, A., Christoffels, V.M. and Kispert, A. (2016). Tbx2 and Tbx3 act downstream of Shh to maintain canonical Wnt signaling during branching morphogenesis of the murine lung. Dev Cell 39: 239-253. PubMed ID: 27720610
Evolutionary Homolog Study:
Numerous signals drive the proliferative expansion of the distal endoderm and the underlying mesenchyme during lung branching morphogenesis, but little is known about how these signals are integrated. By analysis of conditional double mutants, this study shows that the two T-box transcription factor genes Tbx2 and Tbx3 (see Drosophila bi) act together in the lung mesenchyme to maintain branching morphogenesis (see Drosophila trachea development). Expression of both genes depends on epithelially derived Shh (see Drosophila hh) signaling, with additional modulation by Bmp (see Drosophila BMP signaling), Wnt (see Drosophila Wg), and Tgfβ (see Drosophila EGF signaling) signaling. Genetic rescue experiments reveal that Tbx2 and Tbx3 function downstream of Shh to maintain pro-proliferative mesenchymal Wnt signaling, in part by direct repression of the Wnt antagonists Frzb and Shisa3. In combination with previous finding that Tbx2 and Tbx3 repress the cell-cycle inhibitors Cdkn1a and Cdkn1b (see Drosophila dap), this study concludes that Tbx2 and Tbx3 maintain proliferation of the lung mesenchyme by way of at least two molecular mechanisms: regulating cell-cycle regulation and integrating the activity of multiple signaling pathways.

Hu, L., Wang, P., Zhao, R., Li, S., Wang, F., Li, C., Cao, L. and Wu, S. (2016). The Drosophila F-box protein Slimb controls dSmurf protein turnover to regulate the Hippo pathway. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 27856247
SMAD ubiquitination regulatory factors 1 and 2 (Smurf1/2) are members of the HECT domain E3 ligase family which play crucial roles in the regulation of cell cycle progression, planar cell polarity, cancer metastasis and cell apoptosis. It has been previously shown that the Drosophila homolog dSmurf controls the stability of Warts kinase to regulate the Hippo pathway. This study found that the F-box protein Slimb controls dSmurf protein level to regulate the Hippo pathway. Slimb physically associates with dSmurf as revealed by co-immunoprecipitation assay in S2 cells. The C-terminal WD40 repeats of Slimb (188-510 amino acid) and the C-terminal HECT domain of dSmurf (723-1061 amino acid) are necessary for their binding. Interaction with Slimb leads to the ubiquitination and degradation of dSmurf, resulting in negative regulation of dSmurf-mediated Yki phosphorylation and activity in the Hippo pathway. These data reveal a new regulatory mechanism of the Hippo pathway which may provide implications for developing tumor treatment.

Krishnamurthy, V. V., Khamo, J. S., Mei, W., Turgeon, A. J., Ashraf, H. M., Mondal, P., Patel, D. B., Risner, N., Cho, E. E., Yang, J. and Zhang, K. (2016). Reversible optogenetic control of kinase activity during differentiation and embryonic development. Development 143(21): 4085-4094. PubMed ID: 27697903
Evolutionary Homolog Study
Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. This study created an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2; see Drosophila Crytochrome) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proof-of-principle study, an optogenetic Raf kinase (see Drosophila Raf oncogene) was developed that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves light-induced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue light-dependent reversible Raf activation at any desired developmental stage in specific cell lineages. This system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.

Monday, December 5th

Perkins, A. T., Das, T. M., Panzera, L. C. and Bickel, S. E. (2016). Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors. Proc Natl Acad Sci U S A 113: E6823-E6830. PubMed ID: 27791141
One hallmark of aging cells is an increase in oxidative damage caused by reactive oxygen species (ROS). Increased oxidative damage in older oocytes may be one of the factors that leads to premature loss of chromosome cohesion and segregation errors. To test this hypothesis, an RNAi strategy was used to induce oxidative stress in Drosophila oocytes, and the fidelity of chromosome segregation was measured during meiosis. Knockdown of either the cytoplasmic SOD or mitochondrial ROS scavenger superoxide dismutase (SOD) caused a significant increase in segregation errors, and heterozygosity for an smc1 deletion enhanced this phenotype. FISH analysis indicated that SOD knockdown moderately increased the percentage of oocytes with arm cohesion defects. Consistent with premature loss of arm cohesion and destabilization of chiasmata, the frequency at which recombinant homologs missegregate during meiosis I is significantly greater in SOD knockdown oocytes than in controls. Together these results provide an in vivo demonstration that oxidative stress during meiotic prophase induces chromosome segregation errors and support the model that accelerated loss of cohesion in aging human oocytes is caused, at least in part, by oxidative damage.
Johnston, M. J., Bar-Cohen, S., Paroush, Z. and Nystul, T. G. (2016). Phosphorylated Groucho delays differentiation in the follicle stem cell lineage by providing a molecular memory of EGFR signaling in the niche. Development [Epub ahead of print]. PubMed ID: 27836963
In the epithelial follicle stem cells (FSCs) of the Drosophila ovary, Epidermal Growth Factor Receptor (EGFR) signaling promotes self-renewal whereas Notch signaling promotes differentiation of the prefollicle cell (pFC) daughters. Two proteins, Six4 and Groucho (Gro), were identified that link the activity of these two pathways to regulate the earliest cell fate decision in the FSC lineage. The data indicate that Six4 and Gro promote differentiation toward the polar cell fate by promoting Notch pathway activity. This activity of Gro is antagonized by EGFR signaling, which inhibits Gro-dependent repression via p-ERK mediated phosphorylation. The phosphorylated form of Gro persists in newly formed pFCs, which may delay differentiation and provide these cells with a temporary memory of the EGFR signal. Collectively, these findings demonstrate that phosphorylated Gro labels a transition state in the FSC lineage and describe the interplay between Notch and EGFR signaling that governs the differentiation processes during this period.
Gottardo, M., Callaini, G. and Riparbelli, M. G. (2016). Klp10A modulates the localization of centriole-associated proteins during Drosophila male gametogenesis. Cell Cycle [Epub ahead of print] PubMed ID: 27764551
Mutations in Klp10A, a microtubule-depolymerising Kinesin-13, lead to overly long centrioles in Drosophila male germ cells. This study has demonstrated that the loss of Klp10A modifies the distribution of typical proteins involved in centriole assembly and function. In the absence of Klp10A the distribution of Drosophila pericentrin-like protein (Dplp), Sas-4 and Sak/Plk4 that are restricted in control testes to the proximal end of the centriole increase along the centriole length. Remarkably, the cartwheel is lacking or it appears abnormal in mutant centrioles, suggesting that this structure may spatially delimit protein localization. Moreover, the parent centrioles that in control cells have the same dimensions grow at different rates in mutant testes with the mother centrioles longer than the daughters. Daughter centrioles have often an ectopic position with respect to the proximal end of the mothers and failed to recruit Dplp.
Trovisco, V., Belaya, K., Nashchekin, D., Irion, U., Sirinakis, G., Butler, R., Lee, J. J., Gavis, E. R. and St Johnston, D. (2016). bicoid mRNA localises to the Drosophila oocyte anterior by random Dynein-mediated transport and anchoring. Elife 5. PubMed ID: 27791980
bicoid mRNA localises to the Drosophila oocyte anterior from stage 9 of oogenesis onwards to provide a local source for Bicoid protein for embryonic patterning. Live imaging at stage 9 reveals that bicoid mRNA particles undergo rapid Dynein-dependent movements near the oocyte anterior, but with no directional bias. Furthermore, bicoid mRNA localises normally in shot2A2, which abolishes the polarised microtubule organisation. FRAP and photo-conversion experiments demonstrate that the RNA is stably anchored at the anterior, independently of microtubules. Thus, bicoid mRNA is localised by random active transport and anterior anchoring. Super-resolution imaging reveals that bicoid mRNA forms 110-120nm particles with variable RNA content, but constant size. These particles appear to be well-defined structures that package the RNA for transport and anchoring.

Sunday, December 4th

Tofangchi, A., Fan, A. and Saif, M. T. (2016). Mechanism of axonal contractility in embryonic Drosophila motor neurons in vivo. Biophys J 111: 1519-1527. PubMed ID: 27705774
Several in vitro and limited in vivo experiments have shown that neurons maintain a rest tension along their axons intrinsically. They grow in response to stretch but contract in response to loss of tension. This contraction eventually leads to the restoration of the rest tension in axons. However, the mechanism by which axons maintain tension in vivo remains elusive. The objective of this work is to elucidate the key cytoskeletal components responsible for generating tension in axons. Toward this goal, in vivo experiments were conducted on single axons of embryonic Drosophila motor neurons in the presence of various drugs. Each axon was slackened mechanically by bringing the neuromuscular junction toward the central nervous system multiple times. In the absence of any drug, axons shortened and restored the straight configuration within 2-4 min of slackening. The total shortening was approximately 40% of the original length. The recovery rate in each cycle, but not the recovery magnitude, was dependent on the axon's prior contraction history. For example, the contraction time of a previously slackened axon may be twice its first-time contraction. This recovery was significantly hampered with the depletion of ATP, inhibition of myosin motors, and disruption of actin filaments. The disruption of microtubules did not affect the recovery magnitude, but, on the contrary, led to an enhanced recovery rate compared to control cases. These results suggest that the actomyosin machinery is the major active element in axonal contraction, whereas microtubules contribute as resistive/dissipative elements.
Veeranan-Karmegam, R., Boggupalli, D. P., Liu, G. and Gonsalvez, G. B. (2016). A new isoform of Drosophila non-muscle Tropomyosin 1 interacts with Kinesin-1 and functions in oskar mRNA localization. J Cell Sci 129: 4252-4264. PubMed ID: 27802167
Recent studies have revealed that diverse cell types use mRNA localization as a means to establish polarity. Despite the prevalence of this phenomenon, much less is known regarding the mechanism by which mRNAs are localized. The Drosophila melanogaster oocyte provides a useful model for examining the process of mRNA localization. oskar (osk) mRNA is localized at the posterior of the oocyte, thus restricting the expression of Oskar protein to this site. The localization of osk mRNA is microtubule dependent and requires the plus-end-directed motor Kinesin-1. Unlike most Kinesin-1 cargoes, localization of osk mRNA requires the Kinesin heavy chain (Khc) motor subunit, but not the Kinesin light chain (Klc) adaptor. This report, demonstrates that a newly discovered isoform of Tropomyosin 1, referred to as Tm1C, directly interacts with Khc and functions in concert with this microtubule motor to localize osk mRNA. Apart from osk mRNA localization, several additional Khc-dependent processes in the oocyte are unaffected upon loss of Tm1C. These results therefore suggest that the Tm1C-Khc interaction is specific for the osk localization pathway.
Moriwaki, T. and Goshima, G. (2016). Five factors can reconstitute all three phases of microtubule polymerization dynamics. J Cell Biol 215: 357-368. PubMed ID: 27799364
Cytoplasmic microtubules (MTs) undergo growth, shrinkage, and pausing. However, how MT polymerization cycles are produced and spatiotemporally regulated at a molecular level is unclear, as the entire cycle has not been recapitulated in vitro with defined components. In this study, dynamic MT plus end behavior involving all three phases was reconstituted by mixing tubulin with five Drosophila melanogaster proteins (EB1, XMAP215Msps, Sentin, kinesin-13Klp10A, and CLASPMast/Orbit). When singly mixed with tubulin, CLASPMast/Orbit strongly inhibited MT catastrophe and reduced the growth rate. However, in the presence of the other four factors, CLASPMast/Orbit acted as an inducer of pausing. The mitotic kinase Plk1Polo modulated the activity of CLASPMast/Orbit and kinesin-13Klp10A and increased the dynamic instability of MTs, reminiscent of mitotic cells. These results suggest that five conserved proteins constitute the core factors for creating dynamic MTs in cells and that Plk1-dependent phosphorylation is a crucial event for switching from the interphase to mitotic mode.
Teng, X., Qin, L., Le Borgne, R. and Toyama, Y. (2016).. Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium. Development [Epub ahead of print]. PubMed ID: 27888195
Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, the contraction of actomyosin cables formed in apoptotic and neighboring cells drive cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. A model is proposed in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion, and would further influence biochemical signals of neighboring non-apoptotic cells.

Saturday, December 3rd

Khericha, M., Kolenchery, J. B. and Tauber, E. (2016). Neural and non-neural contributions to sexual dimorphism of mid-day sleep in Drosophila melanogaster: a pilot study. Physiol Entomol 41: 327-334. PubMed ID: 27840547
Many of the characteristics associated with mammalian sleep are also observed in Drosophila, making the fruit fly a powerful model organism for studying the genetics of this important process. Included among the similarities is the presence of sexual dimorphic sleep patterns, which, in flies, are manifested as increased mid-day sleep ('siesta') in males compared with females. In the present study, targeted mis-expression of the genes transformer (tra) and tra2 is used to either feminize or masculinize specific neural and non-neural tissues in the fly. Feminization of male flies using three different GAL4 drivers that are expressed in the mushroom bodies induces a female-like reduced siesta, whereas the masculinization of females using these drivers triggers the male-like increased siesta. A similar reversal of sex-specific sleep is also observed by mis-expressing tra in the fat body, which is a key tissue in energy metabolism and hormone secretion. In addition, the daily expression levels of takeout, an important circadian clock output gene, are sexually dimorphic. Taken together, these experiments suggest that sleep sexual dimorphism in D. melanogaster is driven by multiple neural and non-neural circuits, within and outside the brain.
Saltz, J. B. (2016). Genetic variation in social environment construction influences the development of aggressive behavior in Drosophila melanogaster. Heredity (Edinb) [Epub ahead of print]. PubMed ID: 27848947
Individuals are not merely subject to their social environments; they choose and create them, through a process called social environment (or social niche) construction. This study identified multiple mechanisms of social environment construction that differ among natural genotypes of Drosophila melanogaster and investigated their consequences for the development of aggressive behavior. Male genotypes differed in the group sizes that they preferred and in their aggressive behavior; both of these behaviors influenced social experience, demonstrating that these behaviors function as social environment-constructing traits. Further, the effects of social experience-as determined in part by social environment construction-carried over to affect focal male aggression at a later time and with a new opponent. These results provide manipulative experimental support for longstanding hypotheses in psychology, that genetic variation in social environment construction has a causal role in behavioral development. More broadly, these results imply that studies of the genetic basis of complex traits should be expanded to include mechanisms by which genetic variation shapes the environments that individuals experience.
Pavlou, H. J., Lin, A. C., Neville, M. C., Nojima, T., Diao, F., Chen, B. E., White, B. H. and Goodwin, S. F. (2016). Neural circuitry coordinating male copulation. Elife 5. PubMed ID: 27855059
Copulation is the goal of the courtship process, crucial to reproductive success and evolutionary fitness. Identifying the circuitry underlying copulation is a necessary step towards understanding universal principles of circuit operation, and how circuit elements are recruited into the production of ordered action sequences. This study identified key sex-specific neurons that mediate copulation in Drosophila, and define a sexually dimorphic motor circuit in the male abdominal ganglion that mediates the action sequence of initiating and terminating copulation. This sexually dimorphic circuit composed of three neuronal classes - motor neurons, interneurons and mechanosensory neurons - controls the mechanics of copulation. By correlating the connectivity, function and activity of these neurons, the logic was determined for how this circuitry is coordinated to generate this male-specific behavior, and sets the stage for a circuit-level dissection of active sensing and modulation of copulatory behavior.
Kucherenko, M. M., Ilangovan, V., Herzig, B., Shcherbata, H. R. and Bringmann, H. (2016). TfAP-2 is required for night sleep in Drosophila. BMC Neurosci 17: 72. PubMed ID: 27829368
The AP-2 transcription factor APTF-1 is crucially required for developmentally controlled sleep behavior in Caenorhabditis elegans larvae. Its human ortholog, TFAP-2β, causes Char disease and has also been linked to sleep disorders. These data suggest that AP-2 transcription factors may be highly conserved regulators of various types of sleep behavior. This study tested the idea that AP-2 controls adult sleep in Drosophila. Drosophila has one AP-2 ortholog called AP-2, which is essential for viability. To investigate its potential role in sleep behavior and neural development, AP-2 was specifically downregulated in the nervous system. Neuronal AP-2 knockdown almost completely abolished night sleep but did not affect day sleep. AP-2 insufficiency affected nervous system development. Conditional AP-2 knockdown in the adult also produced a modest sleep phenotype, suggesting that AP-2 acts both in larval as well as in differentiated neurons. Thus, these results show that AP-2 transcription factors are highly conserved regulators of development and sleep.

Friday, December 2nd

Kaas, G. A., Kasuya, J., Lansdon, P., Ueda, A., Iyengar, A., Wu, C. F. and Kitamoto, T. (2016). Lithium-responsive seizure-like hyperexcitability is caused by a mutation in the Drosophila voltage-gated Sodium channel gene paralytic. eNeuro 3. PubMed ID: 27844061
Shudderer (Shu) is an X-linked dominant mutation in Drosophila melanogaster identified more than 40 years ago. A previous study showed that Shu caused spontaneous tremors and defects in reactive climbing behavior, and that these phenotypes were significantly suppressed when mutants were fed food containing lithium, a mood stabilizer used in the treatment of bipolar disorder. This unique observation suggested that the Shu mutation affects genes involved in lithium-responsive neurobiological processes. The present study identified Shu as a novel mutant allele of the voltage-gated sodium (Nav) channel gene paralytic (para). Given that hypomorphic para alleles and RNA interference-mediated para knockdown reduced the severity of Shu phenotypes, Shu was classified as a para hypermorphic allele. It was demonstrated that lithium could improve the behavioral abnormalities displayed by other Nav mutants, including a fly model of the human generalized epilepsy with febrile seizures plus. Electrophysiological analysis of Shu showed that lithium treatment did not acutely suppress Nav channel activity, indicating that the rescue effect of lithium resulted from chronic physiological adjustments to this drug. Microarray analysis revealed that lithium significantly alters the expression of various genes in Shu, including those involved in innate immune responses, amino acid metabolism, and oxidation-reduction processes, raising the interesting possibility that lithium-induced modulation of these biological pathways may contribute to such adjustments. Overall, these findings demonstrate that Nav channel mutants in Drosophila are valuable genetic tools for elucidating the effects of lithium on the nervous system in the context of neurophysiology and behavior.
Kinghorn, K. J., Gronke, S., Castillo-Quan, J. I., Woodling, N. S., Li, L., Sirka, E., Gegg, M., Mills, K., Hardy, J., Bjedov, I. and Partridge, L. (2016). A Drosophila model of Neuronopathic Gaucher Disease demonstrates lysosomal-autophagic defects and altered mTOR signalling and is functionally rescued by rapamycin. J Neurosci 36: 11654-11670. PubMed ID: 27852774
Glucocerebrosidase (GBA1) mutations are associated with Gaucher disease (GD), an autosomal recessive disorder caused by functional deficiency of glucocerebrosidase (GBA), a lysosomal enzyme that hydrolyzes glucosylceramide to ceramide and glucose. Neuronopathic forms of GD can be associated with rapid neurological decline (Type II) or manifest as a chronic form (Type III) with a wide spectrum of neurological signs. Furthermore, there is now a well-established link between GBA1 mutations and Parkinson's disease (PD), with heterozygote mutations in GBA1 considered the commonest genetic defect in PD. This study describes a novel Drosophila model of GD that lacks the two fly GBA1 orthologs (Gba1a and Gba1b). This knock-out model recapitulates the main features of GD at the cellular level with severe lysosomal defects and accumulation of glucosylceramide in the fly brain. A block in autophagy flux was demonstrated in association with reduced lifespan, age-dependent locomotor deficits and accumulation of autophagy substrates in dGBA-deficient fly brains. Furthermore, mechanistic target of rapamycin (mTOR) signaling is downregulated in dGBA knock-out flies, with a concomitant upregulation of Mitf gene expression, the fly ortholog of mammalian TFEB, likely as a compensatory response to the autophagy block. Moreover, the mTOR inhibitor rapamycin is able to partially ameliorate the lifespan, locomotor, and oxidative stress phenotypes. Together, these results demonstrate that this dGBA1-deficient fly model is a useful platform for the further study of the role of lysosomal-autophagic impairment and the potential therapeutic benefits of rapamycin in neuronopathic GD. These results also have important implications for the role of autophagy and mTOR signaling in GBA1-associated PD.
Kumar, S., Jang, I. H., Kim, C. W., Kang, D. W., Lee, W. J. and Jo, H. (2016). Functional screening of mammalian mechanosensitive genes using Drosophila RNAi library- Smarcd3/Bap60 is a mechanosensitive pro-inflammatory gene. Sci Rep 6: 36461. PubMed ID: 27819340
Disturbed blood flow (d-flow) induces atherosclerosis by altering the expression of mechanosensitive genes in the arterial endothelium. Previous studies have identified >580 mechanosensitive genes in the mouse arterial endothelium, but their role in endothelial inflammation is incompletely understood. From this set, 84 Drosophila RNAi lines were obtained that silence the target gene under the control of upstream activation sequence (UAS) promoter. These lines were crossed with C564-GAL4 flies expressing GFP under the control of drosomycin promoter, an NF-κB target gene and a marker of pathogen-induced inflammation. Silencing of psmd12 or ERN1 decreased infection-induced drosomycin expression, while Bap60 silencing significantly increased the drosomycin expression. Interestingly, knockdown of Bap60 in adult flies using temperature-inducible Bap60 RNAi enhanced drosomycin expression upon Gram-positive bacterial challenge but the basal drosomycin expression remained unchanged compared to the control. In the mammalian system, smarcd3 (mammalian ortholog of Bap60) expression was reduced in the human- and mouse aortic endothelial cells exposed to oscillatory shear in vitro as well as in the d-flow regions of mouse arterial endothelium in vivo. Moreover, siRNA-mediated knockdown of smarcd3 induced endothelial inflammation. In summary, an in vivo Drosophila RNAi screening method identified flow-sensitive genes that regulate endothelial inflammation.
Vianna, M. C., Poleto, D. C., Gomes, P. F., Valente, V. and Paco-Larson, M. L. (2016). Drosophila ataxin-2 gene encodes two differentially expressed isoforms and its function in larval fat body is crucial for development of peripheral tissues. FEBS Open Bio 6: 1040-1053. PubMed ID: 27833845
Different isoforms of ataxin-2 are predicted in Drosophila and may underlie different cellular processes. This study validated the isoforms B and C of Drosophila ataxin-2 locus (dAtx2), which was found to be expressed in various tissues and at different levels during development. dAtx2-B mRNA was detected at low amounts during all developmental stages, whereas dAtx2-C mRNA levels increase by eightfold from L3 to pupal-adult stages. Higher amounts of dAtx2-B protein were detected in embryos, while dAtx2-C protein was also expressed in higher levels in pupal-adult stages, indicating post-transcriptional control for isoform B and transcription induction for isoform C, respectively. Moreover, in the fat body of L3 larvae dAtx2-C, but not dAtx2-B, accumulates in cytoplasmic foci that colocalize with sec23, a marker of endoplasmic reticulum exit sites (ERES). Interestingly, animals subjected to selective knockdown of dAtx2 in the larval fat body do not complete metamorphosis and die at the third larval stage or early puparium. Additionally, larvae knocked down for dAtx2, grown at 29 ° C, are significantly smaller than control animals due to reduction in DNA replication and cell growth, consistent with the decreased levels of phosphorylated-AKT observed in the fat body. Based on the localization of ataxin-2 (dAtx2-C) in ERESs, and on the phenotypes observed by dAtx2 knockdown in the larval fat body, a possible role for this protein in processes that regulate ERES formation is suggested. These data provide new insights into the biological function of ataxin-2 with potential relevance to neurodegenerative diseases.

Thursday, December 1st

Sizemore, T. R. and Dacks, A. M. (2016). Serotonergic modulation differentially targets distinct network elements within the antennal lobe of Drosophila melanogaster. Sci Rep 6: 37119. PubMed ID: 27845422
Neuromodulation confers flexibility to anatomically-restricted neural networks so that animals are able to properly respond to complex internal and external demands. However, determining the mechanisms underlying neuromodulation is challenging without knowledge of the functional class and spatial organization of neurons that express individual neuromodulatory receptors. This study describes the number and functional identities of neurons in the antennal lobe of Drosophila melanogaster that express each of the receptors for one such neuromodulator, serotonin (5-HT). Although 5-HT enhances odor-evoked responses of antennal lobe projection neurons (PNs) and local interneurons (LNs), the receptor basis for this enhancement is unknown. Endogenous reporters of transcription and translation for each of the five 5-HT receptors (5-HTRs) were used to identify neurons, based on cell class and transmitter content, that express each receptor. Specific receptor types are expressed by distinct combinations of functional neuronal classes. For instance, the excitatory PNs express the excitatory 5-HTRs (5-HT2 type and 5-HT7), the 5-HT1 type receptors are generally inhibitory, and distinct classes of LNs each express different 5-HTRs. This study therefore provides a detailed atlas of 5-HT receptor expression within a well-characterized neural network, and enables future dissection of the role of serotonergic modulation of olfactory processing.
Shimada, N., Inami, S., Sato, S., Kitamoto, T. and Sakai, T. (2016). Modulation of light-driven arousal by LIM-homeodomain transcription factor Apterous in large PDF-positive lateral neurons of the Drosophila brain. Sci Rep 6: 37255. PubMed ID: 27853240
Apterous (Ap), the best studied LIM-homeodomain transcription factor in Drosophila, cooperates with the cofactor Chip (Chi) to regulate transcription of specific target genes. Although Ap regulates various developmental processes, its function in the adult brain remains unclear. This study reports that Ap and Chi in the neurons expressing PDF, a neuropeptide, play important roles in proper sleep/wake regulation in adult flies. PDF-expressing neurons consist of two neuronal clusters: small ventral-lateral neurons (s-LNvs) acting as the circadian pacemaker and large ventral-lateral neurons (l-LNvs) regulating light-driven arousal. Ap localizes to the nuclei of s-LNvs and l-LNvs. In light-dark (LD) cycles, RNAi knockdown or the targeted expression of dominant-negative forms of Ap or Chi in PDF-expressing neurons or l-LNvs promoted arousal. In contrast, in constant darkness, knockdown of Ap in PDF-expressing neurons did not promote arousal, indicating that a reduced Ap function in PDF-expressing neurons promotes light-driven arousal. Furthermore, Ap expression in l-LNvs showed daily rhythms (peaking at midnight), which are generated by a direct light-dependent mechanism rather than by the endogenous clock. These results raise the possibility that the daily oscillation of Ap expression in l-LNvs may contribute to the buffering of light-driven arousal in wild-type flies.
Schlegel, P., Texada, M. J., Miroschnikow, A., Schoofs, A., Huckesfeld, S., Peters, M., Schneider-Mizell, C. M., Lacin, H., Li, F., Fetter, R. D., Truman, J. W., Cardona, A. and Pankratz, M. J. (2016). Synaptic transmission parallels neuromodulation in a central food-intake circuit. Elife 5 [Epub ahead of print]. PubMed ID: 27845623
NeuromedinU is a potent regulator of food intake and activity in mammals. In Drosophila, neurons producing the homologous neuropeptide hugin regulate feeding and locomotion in a similar manner. This study used EM-based reconstruction to generate the entire connectome of hugin-producing neurons in the Drosophila larval CNS. Hugin neurons were shown to use synaptic transmission in addition to peptidergic neuromodulation, and acetylcholine was identified as a key transmitter. Hugin neuropeptide and acetylcholine are both necessary for the regulatory effect on feeding. Subtypes of hugin neurons connect chemosensory to endocrine system by combinations of synaptic and peptide-receptor connections. Targets include endocrine neurons producing DH44, a CRH-like peptide, and insulin-like peptides. Homologs of these peptides are likewise downstream of neuromedinU, revealing striking parallels in flies and mammals. It is proposed that hugin neurons are part of an ancient physiological control system that has been conserved at functional and molecular level.
Suver, M. P., Huda, A., Iwasaki, N., Safarik, S. and Dickinson, M. H. (2016). An array of descending visual interneurons encoding self-motion in Drosophila. J Neurosci 36: 11768-11780. PubMed ID: 27852783
The means by which brains transform sensory information into coherent motor actions is poorly understood. In flies, a relatively small set of descending interneurons are responsible for conveying sensory information and higher-order commands from the brain to motor circuits in the ventral nerve cord. This study describes three pairs of genetically identified descending interneurons that integrate information from wide-field visual interneurons and project directly to motor centers controlling flight behavior. The physiological responses of these three cells were measured during flight, and they were found to respond maximally to visual movement corresponding to rotation around three distinct body axes. After characterizing the tuning properties of an array of nine putative upstream visual interneurons, it was shown that simple linear combinations of their outputs can predict the responses of the three descending cells. Last, a machine vision-tracking system was developed that allows monitoring of multiple motor systems simultaneously, and each visual descending interneuron class was found to correlate with a discrete set of motor programs.

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