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


Thursday August 31st, 2017

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Chen, D., Sitaraman, D., Chen, N., Jin, X., Han, C., Chen, J., Sun, M., Baker, B. S., Nitabach, M. N. and Pan, Y. (2017). Genetic and neuronal mechanisms governing the sex-specific interaction between sleep and sexual behaviors in Drosophila. Nat Commun 8(1): 154. PubMed ID: 28754889
Animals execute one particular behavior among many others in a context-dependent manner, yet the mechanisms underlying such behavioral choice remain poorly understood. This research studied how two fundamental behaviors, sex and sleep, interact at genetic and neuronal levels in Drosophila. An increased need for sleep was shown to inhibits male sexual behavior by decreasing the activity of the male-specific P1 neurons that coexpress the sex determination genes fruM and dsx, but does not affect female sexual behavior. Further, a sex-specific neuronal circuit was delineated wherein the P1 neurons encoding increased courtship drive suppressed male sleep by forming mutually excitatory connections with the fruM -positive sleep-controlling DN1 neurons. In addition, FRUM was found to regulates male courtship and sleep through distinct neural substrates. These studies reveal the genetic and neuronal basis underlying the sex-specific interaction between sleep and sexual behaviors in Drosophila, and provide insights into how competing behaviors are co-regulated. Genes and circuits involved in sleep and sexual arousal have been extensively studied in Drosophila. This study has identified the sex determination genes fruitless and doublesex, and a sex-specific P1-DN1 neuronal feedback that governs the interaction between these competing behaviors.
Corthals, K., Heukamp, A. S., Kossen, R., Grosshennig, I., Hahn, N., Gras, H., Gopfert, M. C., Heinrich, R. and Geurten, B. R. H. (2017). Neuroligins Nlg2 and Nlg4 affect social behavior in Drosophila melanogaster. Front Psychiatry 8: 113. PubMed ID: 28740469
The genome of Drosophila melanogaster includes homologs to approximately one-third of the currently known human disease genes. Flies and humans share many biological processes, including the principles of information processing by excitable neurons, synaptic transmission, and the chemical signals involved in intercellular communication. Studies on the molecular and behavioral impact of genetic risk factors of human neuro-developmental disorders [autism spectrum disorders (ASDs), schizophrenia, attention deficit hyperactivity disorders, and Tourette syndrome] increasingly use the well-studied social behavior of D. melanogaster, an organism that is amenable to a large variety of genetic manipulations. Neuroligins (Nlgs) are a family of phylogenetically conserved postsynaptic adhesion molecules present (among others) in nematodes, insects, and mammals. Impaired function of Nlgs (particularly of Nlg 3 and 4) has been associated with ASDs in humans and impaired social and communication behavior in mice. Making use of a set of behavioral and social assays, this study analyzed the impact of two Drosophila Nlgs, Dnlg2 and Dnlg4, which are differentially expressed at excitatory and inhibitory central nervous synapses, respectively. Both Nlgs seem to be associated with diurnal activity and social behavior. Even though deficiencies in Dnlg2 and Dnlg4 appeared to have no effects on sensory or motor systems, they differentially impacted on social interactions, suggesting that social behavior is distinctly regulated by these Nlgs.
von Reyn, C. R., Nern, A., Williamson, W. R., Breads, P., Wu, M., Namiki, S. and Card, G. M. (2017). Feature integration drives probabilistic behavior in the Drosophila escape response. Neuron 94(6): 1190-1204.e1196. PubMed ID: 28641115
Animals rely on dedicated sensory circuits to extract and encode environmental features. How individual neurons integrate and translate these features into behavioral responses remains a major question. This study has identified a visual projection neuron type that conveys predator approach information to the Drosophila giant fiber (GF) escape circuit. Genetic removal of this input during looming stimuli reveals that it encodes angular expansion velocity, whereas other input cell type(s) encode angular size. Motor program selection and timing emerge from linear integration of these two features within the GF. Linear integration improves size detection invariance over prior models and appropriately biases motor selection to rapid, GF-mediated escapes during fast looms. These findings suggest feature integration, and motor control may occur as simultaneous operations within the same neuron and establish the Drosophila escape circuit as a model system in which these computations may be further dissected at the circuit level.
Yun, L., Chen, P. J., Singh, A., Agrawal, A. F. and Rundle, H. D. (2017). The physical environment mediates male harm and its effect on selection in females. Proc Biol Sci 284(1858). PubMed ID: 28679725
Recent experiments indicate that male preferential harassment of high-quality females reduces the variance in female fitness, thereby weakening natural selection through females and hampering adaptation and purging. It is proposed that this phenomenon, which results from a combination of male choice and male-induced harm, should be mediated by the physical environment in which intersexual interactions occur. Using Drosophila melanogaster, this study examined intersexual interactions in small and simple (standard fly vials) versus slightly more realistic (small cages with spatial structure) environments. In these more realistic environments, sexual interactions are less frequent, are no longer biased towards high-quality females, and that overall male harm is reduced. Next, the selective advantage of high- over low-quality females was examined while manipulating the opportunity for male choice. Male choice weakens the viability advantage of high-quality females in the simple environment, consistent with previous work, but strengthens selection on females in the more realistic environment. Laboratory studies in simple environments have strongly shaped our understanding of sexual conflict but may provide biased insight. These results suggest that the physical environment plays a key role in the evolutionary consequences of sexual interactions and ultimately the alignment of natural and sexual selection.

Wednesday, August 30th

Chung, C. G., Kwon, M. J., Jeon, K. H., Hyeon, D. Y., Han, M. H., Park, J. H., Cha, I. J., Cho, J. H., Kim, K., Rho, S., Kim, G. R., Jeong, H., Lee, J. W., Kim, T., Kim, K., Kim, K. P., Ehlers, M. D., Hwang, D. and Lee, S. B. (2017). Golgi outpost synthesis impaired by toxic polyglutamine proteins contributes to dendritic pathology in neurons. Cell Rep 20(2): 356-369. PubMed ID: 28700938
Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. This study shows that nuclear polyglutamine (polyQ) toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs) and a decreased supply of plasma membrane (PM) in Drosophila class IV dendritic arborization (da) (C4 da) neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP)-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP), which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, these results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway.
Tao, J., Bulgari, D., Deitcher, D. L. and Levitan, E. S. (2017). Limited distal organelles and synaptic function in extensive monoaminergic innervation. J Cell Sci [Epub ahead of print]. PubMed ID: 28600320
Organelles such as neuropeptide-containing dense-core vesicles (DCVs) and mitochondria travel down axons to supply synaptic boutons. DCV distribution among en passant boutons in small axonal arbors is mediated by circulation with bidirectional capture. However, it is not known how organelles are distributed in extensive arbors associated with volume transmission and neuromodulation by monoamines and neuropeptides and mammalian dopamine neuron vulnerability. Therefore, this study examined presynaptic organelle distribution in Drosophila octopamine neurons that innervate approximately 20 muscles with approximately 1500 boutons. Unlike in smaller arbors, distal boutons in these arbors contain fewer DCVs and mitochondria, although active zones are present. Absence of vesicle circulation is evident by proximal nascent DCV delivery, limited impact of retrograde transport and older distal DCVs. Traffic studies show that DCV axonal transport and synaptic capture are not scaled for extensive innervation, thus limiting distal delivery. Activity-induced synaptic endocytosis and synaptic neuropeptide release are also reduced distally. It is proposed that limits in organelle transport and synaptic capture compromise distal synapse maintenance and function in extensive axonal arbors, thereby affecting development, plasticity and vulnerability to neurodegenerative disease.
Rui, M., Qian, J., Liu, L., Cai, Y., Lv, H., Han, J., Jia, Z. and Xie, W. (2017). The neuronal protein Neurexin directly interacts with the Scribble-Pix complex to stimulate F-actin assembly for synaptic vesicle clustering. J Biol Chem [Epub ahead of print]. PubMed ID: 28710284
Synaptic vesicles (SVs) form distinct pools at synaptic terminals. However, how SV cluster in particular synaptic compartments to maintain normal neurotransmitter release remains a mystery. The presynaptic protein Neurexin (NRX) plays a significant role in synaptic architecture and function. However, the role of NRX in SV clustering is unclear. Using the neuromuscular junction at the 2-3 instar stages of Drosophila larvae as a model and biochemical, imaging, and electrophysiology techniques, this study demonstrated that Drosophila NRX (DNRX) plays critical roles in regulating synaptic terminal clustering and release of SVs. We found that DNRX controls the terminal clustering and release of SVs by stimulating presynaptic F-actin. Furthermore, the results indicate that DNRX functions through the scaffold protein Scribble and the GEF protein DPix to activate the small GTPase Rac1. A direct interaction was observed between the C-terminal PDZ-binding motif of DNRX and the PDZ domains of Scribble, and Scribble bridges DNRX to DPix, forming a DNRX/Scribble/DPix complex that activates Rac1 and subsequently stimulates presynaptic F-actin assembly and SV clustering. Taken together, this work provides important insights into the function of DNRX in regulating SV clustering, which could help inform further research into pathological neurexin-mediated mechanisms in neurological disorders such as autism.
Chamberland, S., Yang, H. H., Pan, M. M., Evans, S. W., Guan, S., Chavarha, M., Yang, Y., Salesse, C., Wu, H., Wu, J. C., Clandinin, T. R., Toth, K., Lin, M. Z. and St-Pierre, F. (2017). Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators. Elife 6. PubMed ID: 28749338
Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits, but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. This study reports ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, robust single-trial detection of action potentials was demonstrated in organotypic slice cultures. It was also shown that ASAP2s enables two-photon imaging of graded potentials with subcellular resolution in organotypic slice cultures and in Drosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.

Tuesday, August 29th

Choi, S., Quan, X., Bang, S., Yoo, H., Kim, J., Park, J., Park, K. S. and Chung, J. (2017). Mitochondrial calcium uniporter in Drosophila transfers calcium between the endoplasmic reticulum and mitochondria in oxidative stress-induced cell death. J Biol Chem. PubMed ID: 28726639
Mitochondrial calcium plays critical roles in diverse cellular processes ranging from energy metabolism to cell death. Previous studies have demonstrated that mitochondrial calcium uptake is mainly mediated by the mitochondrial calcium uniporter (MCU) complex. However, the roles of the MCU complex in calcium transport, signaling, and dysregulation by oxidative stress still remain unclear. This study confirmed that Drosophila MCU contains evolutionarily conserved structures and requires essential MCU regulator (EMRE) for its calcium channel activities. Drosophila MCU loss-of-function mutants, which lacked mitochondrial calcium uptake in response to caffeine stimulation, were generated. Basal metabolic activities were not significantly affected in these MCU mutants as observed in examinations of body weight, food intake, body sugar level, and starvation-induced autophagy. However, oxidative stress-induced increases in mitochondrial calcium, mitochondrial membrane potential depolarization, and cell death were prevented in these mutants. It was also found that inositol 1,4,5-trisphosphate receptor (IP3R) genetically interacts with Drosophila MCU and effectively modulates mitochondrial calcium uptake upon oxidative stress. Taken together, these results support the idea that Drosophila MCU is responsible for ER-to-mitochondrial calcium transfer and for cell death due to mitochondrial dysfunction under oxidative stress.
Kim, T. S., Shin, Y. H., Lee, H. M., Kim, J. K., Choe, J. H., Jang, J. C., Um, S., Jin, H. S., Komatsu, M., Cha, G. H., Chae, H. J., Oh, D. C. and Jo, E. K. (2017). Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway. Sci Rep 7(1): 3431. PubMed ID: 28611371
The induction of host cell autophagy by various autophagy inducers contributes to the antimicrobial host defense against Mycobacterium tuberculosis (Mtb), a major pathogenic strain that causes human tuberculosis. This study presents a role for the newly identified cyclic peptides ohmyungsamycins (OMS) A and B in the antimicrobial responses against Mtb infections by activating autophagy in murine bone marrow-derived macrophages (BMDMs). OMS robustly activated autophagy, which was essentially required for the colocalization of LC3 autophagosomes with bacterial phagosomes and antimicrobial responses against Mtb in BMDMs. Using a Drosophila melanogaster-Mycobacterium marinum infection model, this study shows that OMS-A-induced autophagy contributed to the increased survival of infected flies and the limitation of bacterial load. It was further shown that OMS triggered AMP-activated protein kinase (AMPK) activation, which was required for OMS-mediated phagosome maturation and antimicrobial responses against Mtb. Moreover, treating BMDMs with OMS led to dose-dependent inhibition of macrophage inflammatory responses, which was also dependent on AMPK activation. Collectively, these data show that OMS is a promising candidate for new anti-mycobacterial therapeutics by activating antibacterial autophagy via AMPK-dependent signaling and suppressing excessive inflammation during Mtb infections.
Lin, L., Rodrigues, F., Kary, C., Contet, A., Logan, M., Baxter, R. H. G., Wood, W. and Baehrecke, E. H. (2017). Complement-related regulates autophagy in neighboring cells. Cell 170(1): 158-171.e158. PubMed ID: 28666117
Autophagy degrades cytoplasmic components and is important for development and human health. Although autophagy is known to be influenced by systemic intercellular signals, the proteins that control autophagy are largely thought to function within individual cells. This study reports that Drosophila macroglobulin complement-related (Mcr), a complement ortholog, plays an essential role during developmental cell death and inflammation by influencing autophagy in neighboring cells. This function of Mcr involves the immune receptor Draper, suggesting a relationship between autophagy and the control of inflammation. Interestingly, Mcr function in epithelial cells is required for macrophage autophagy and migration to epithelial wounds, a Draper-dependent process. This study reveals, unexpectedly, that complement-related from one cell regulates autophagy in neighboring cells via an ancient immune signaling program.
An, P. N. T., Shimaji, K., Tanaka, R., Yoshida, H., Kimura, H., Fukusaki, E. and Yamaguchi, M. (2017). Epigenetic regulation of starvation-induced autophagy in Drosophila by histone methyltransferase G9a. Sci Rep 7(1): 7343. PubMed ID: 28779125
Epigenetics is now emerging as a key regulation in response to various stresses. This study identified the Drosophila histone methyltransferase G9a (dG9a) as a key factor to acquire tolerance to starvation stress. The depletion of dG9a led to high sensitivity to starvation stress in adult flies, while its overexpression induced starvation stress resistance. The catalytic domain of dG9a was not required for starvation stress resistance. dG9a plays no apparent role in tolerance to other stresses including heat and oxidative stresses. Metabolomic approaches were applied to investigate global changes in the metabolome due to the loss of dG9a during starvation stress. The results obtained indicated that dG9a plays an important role in maintaining energy reservoirs including amino acid, trehalose, glycogen, and triacylglycerol levels during starvation. Further investigations on the underlying mechanisms showed that the depletion of dG9a repressed starvation-induced autophagy by controlling the expression level of Atg8a, a critical gene for the progression of autophagy, in a different manner to that in cancer cells. These results indicate a positive role for dG9a in starvation-induced autophagy.

Monday August 29th

Chen, J., Xue, J., Ruan, J., Zhao, J., Tang, B. and Duan, R. (2017). Drosophila CHIP protects against mitochondrial dysfunction by acting downstream of Pink1 in parallel with Parkin. FASEB J. [Epub ahead of print] PubMed ID: 28778978
Mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin function in a common pathway to regulate mitochondrial homeostasis contributing to the pathogenesis of Parkinson disease. The carboxyl terminus of Hsc70-interacting protein (CHIP) acts as a heat shock protein 70/heat shock protein 90 cochaperone to mediate protein folding or as an E3 ubiquitin ligase to target proteins for degradation. In this study, overexpression of Drosophila CHIP suppressed a range of Pink1 mutant phenotypes in flies, including abnormal wing posture, thoracic indentation, locomotion defects, muscle degeneration, and loss of dopaminergic neurons. Mitochondrial defects of Pink1 mutant, such as excessive fusion, reduced ATP content, and crista disorganization, were rescued by CHIP but not its ligase-dead mutants. Similar phenotypes and mitochondrial impairment were ameliorated in Parkin mutant flies by wild-type CHIP. Inactivation of CHIP with null fly mutants resulted in mitochondrial defects, such as reduced thoracic ATP content at 3 d old, decreased thoracic mitochondrial DNA content, and defective mitochondrial morphology at 60 d old. CHIP mutants did not exacerbate the phenotypes of Pink1 mutant flies but markedly shortened the life span of Parkin mutant flies. These results indicate that CHIP is involved in mitochondrial integrity and may act downstream of Pink1 in parallel with Parkin.
Agrawal, P., Houl, J. H., Gunawardhana, K. L., Liu, T., Zhou, J., Zoran, M. J. and Hardin, P. E. (2017). Drosophila CRY entrains clocks in body tissues to light and maintains passive membrane properties in a non-clock body tissue independent of light. Curr Biol [Epub ahead of print]. PubMed ID: 28781048
Circadian clocks regulate daily rhythms in physiology, metabolism, and behavior via cell-autonomous transcriptional feedback loops. In Drosophila, the blue-light photoreceptor Cryptrochrome (CRY) synchronizes these feedback loops to light:dark cycles by binding to and degrading Timeless (TIM) protein. CRY also acts independently of TIM in Drosophila to alter potassium channel conductance in arousal neurons after light exposure, and in many animals CRY acts independently of light to repress rhythmic transcription. CRY expression has been characterized in the Drosophila brain and eyes, but not in peripheral clock and non-clock tissues in the body. To investigate CRY expression and function in body tissues, a GFP-tagged-cry transgene was generated that rescues light-induced behavioral phase resetting in cry03 mutant flies and sensitively reports GFP-CRY expression. In bodies, CRY is detected in clock-containing tissues including Malpighian tubules, where it mediates both light-dependent TIM degradation and clock function. In larval salivary glands, which lack clock function but are amenable to electrophysiological recording, CRY prevents membrane input resistance from falling to low levels in a light-independent manner. The ability of CRY to maintain high input resistance in these non-excitable cells also requires the K+ channel subunits Hyperkinetic, Shaker, and ether-a-go-go. These findings for the first time define CRY expression in Drosophila peripheral tissues and reveal that CRY acts together with K+ channels to maintain passive membrane properties in a non-clock-containing peripheral tissue independent of light.
Wu, B., Li, J., Chou, Y. H., Luginbuhl, D. and Luo, L. (2017). Fibroblast growth factor signaling instructs ensheathing glia wrapping of Drosophila olfactory glomeruli. Proc Natl Acad Sci U S A. PubMed ID: 28674010
The formation of complex but highly organized neural circuits requires interactions between neurons and glia. During the assembly of the Drosophila olfactory circuit, 50 olfactory receptor neuron (ORN) classes and 50 projection neuron (PN) classes form synaptic connections in 50 glomerular compartments in the antennal lobe, each of which represents a discrete olfactory information-processing channel. Each compartment is separated from the adjacent compartments by membranous processes from ensheathing glia. This study shows that Thisbe, an FGF released from olfactory neurons, particularly from local interneurons, instructs ensheathing glia to wrap each glomerulus. The Heartless FGF receptor acts cell-autonomously in ensheathing glia to regulate process extension so as to insulate each neuropil compartment. Overexpressing Thisbe in ORNs or PNs causes overwrapping of the glomeruli their axons or dendrites target. Failure to establish the FGF-dependent glia structure disrupts precise ORN axon targeting and discrete glomerular formation.
Cardoso, M. A., Fontenele, M., Lim, B., Bisch, P. M., Shvartsman, S. Y. and Araujo, H. M. (2017). A novel function for the IkappaB inhibitor Cactus in promoting Dorsal nuclear localization and activity in the Drosophila embryo. Development 144(16): 2907-2913. PubMed ID: 28705899
The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. This study investigated how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NFκB inhibitor, ensuring a correct response to Toll signals.

Sunday, August 27th

Strom, A. R., Emelyanov, A. V., Mir, M., Fyodorov, D. V., Darzacq, X. and Karpen, G. H. (2017). Phase separation drives heterochromatin domain formation. Nature [Epub ahead of print]. PubMed ID: 28636597
Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability, and silencing of transposon and gene expression. Heterochromatin is highly enriched for repetitive sequences, and is defined epigenetically by methylation of histone H3 at lysine 9 and recruitment of its binding partner heterochromatin protein 1 (HP1). This study presents data that support the hypothesis that the formation of heterochromatin domains is mediated by phase separation, a phenomenon that gives rise to diverse non-membrane-bound nuclear, cytoplasmic and extracellular compartments. Drosophila HP1a protein is shown to undergoes liquid-liquid demixing in vitro, and nucleates into foci that display liquid properties during the first stages of heterochromatin domain formation in early Drosophila embryos. Furthermore, in both Drosophila and mammalian cells, heterochromatin domains exhibit dynamics that are characteristic of liquid phase-separation, including sensitivity to the disruption of weak hydrophobic interactions, and reduced diffusion, increased coordinated movement and inert probe exclusion at the domain boundary. It is concluded that heterochromatic domains form via phase separation, and mature into a structure that includes liquid and stable compartments. It is proposed that emergent biophysical properties associated with phase-separated systems are critical to understanding the unusual behaviours of heterochromatin, and how chromatin domains in general regulate essential nuclear functions.
Chavez, J., Murillo-Maldonado, J. M., Bahena, V., Cruz, A. K., Castaneda-Sortibran, A., Rodriguez-Arnaiz, R., Zurita, M. and Valadez-Graham, V. (2017). dAdd1 and dXNP prevent genome instability by maintaining HP1a localization at Drosophila telomeres. Chromosoma [Epub ahead of print]. PubMed ID: 28688038
Telomeres are important contributors to genome stability, as they prevent linear chromosome end degradation and contribute to the avoidance of telomeric fusions. An important component of the telomeres is the heterochromatin protein 1a (HP1a). Mutations in Su(var)205, the gene encoding HP1a in Drosophila, result in telomeric fusions, retrotransposon regulation loss and larger telomeres, leading to chromosome instability. Previously, it was found that several proteins physically interact with HP1a, including dXNP and dAdd1 (orthologues to the mammalian ATRX gene). This study found that mutations in the genes encoding the dXNP and dAdd1 proteins affect chromosome stability, causing chromosomal aberrations, including telomeric defects, similar to those observed in Su(var)205 mutants. In somatic cells, dXNP and dAdd1 were observed to participate in the silencing of the telomeric HTT array of retrotransposons, preventing anomalous retrotransposon transcription and integration. Furthermore, the lack of dAdd1 results in the loss of HP1a from the telomeric regions without affecting other chromosomal HP1a binding sites; mutations in dxnp also affected HP1a localization but not at all telomeres, suggesting a specialized role for dAdd1 and dXNP proteins in locating HP1a at the tips of the chromosomes. These results place dAdd1 as an essential regulator of HP1a localization and function in the telomere heterochromatic domain.
Colmenares, S. U., Swenson, J. M., Langley, S. A., Kennedy, C., Costes, S. V. and Karpen, G. H. (2017). Drosophila histone demethylase KDM4A has enzymatic and non-enzymatic roles in controlling heterochromatin integrity. Dev Cell 42(2): 156-169.e155. PubMed ID: 28743002
Eukaryotic genomes are broadly divided between gene-rich euchromatin and the highly repetitive heterochromatin domain, which is enriched for proteins critical for genome stability and transcriptional silencing. This study shows that Drosophila KDM4A (dKDM4A), previously characterized as a euchromatic histone H3 K36 demethylase and transcriptional regulator, predominantly localizes to heterochromatin and regulates heterochromatin position-effect variegation (PEV), organization of repetitive DNAs, and DNA repair. dKDM4A demethylase activity is dispensable for PEV. In contrast, dKDM4A enzymatic activity is required to relocate heterochromatic double-strand breaks outside the domain, as well as for organismal survival when DNA repair is compromised. Finally, DNA damage triggers dKDM4A-dependent changes in the levels of H3K56me3, suggesting that dKDM4A demethylates this heterochromatic mark to facilitate repair. It is concluded that dKDM4A, in addition to its previously characterized role in euchromatin, utilizes both enzymatic and structural mechanisms to regulate heterochromatin organization and functions.
Tang, X., Cao, J., Zhang, L., Huang, Y., Zhang, Q. and Rong, Y. S. (2017). Maternal Haploid, a metalloprotease enriched at the largest satellite repeat and essential for genome integrity in Drosophila embryos. Genetics [Epub ahead of print]. PubMed ID: 28615282
The incorporation of the paternal genome into the zygote during fertilization requires chromatin remodeling. The maternal haploid (mh) mutation in Drosophila affects this process and leads to the formation of haploid embryos without the paternal genome. mh encodes the Drosophila homolog of SPRTN, a conserved protease essential for resolving DNA-protein cross-linked products. This study characterized the role of MH in genome maintenance. It is not understood how MH protects the paternal genome during fertilization particularly in lights of the finding that MH is present in both parental pro-nuclei during zygote formation. Maternal chromosomes in mh-mutant embryos were shown to experience instabilities in the absence of the paternal genome, which suggests that MH is generally required for chromosome stability during embryogenesis. This is consistent with the finding that MH is abundantly present on chromatin throughout the cell cycle. Remarkably, MH is prominently enriched at the 359bp satellite repeats during interphase, which becomes unstable without MH. This dynamic localization and specific enrichment of MH at the 359 repeats resemble that of Topoisomerase 2 (Top2), suggesting that MH regulates Top2 possibly as a protease for the resolution of Top2-DNA intermediates. It is proposed that maternal MH removes proteins specifically enriched on sperm chromatin. In the absence of that function, paternal chromosomes are precipitously lost. This mode of paternal chromatin remodeling is likely conserved and the unique phenotype of the Drosophila mh mutants represents a rare opportunity to gain insights into the process that has been difficult to study.

Saturday, August 26th

Beckwith, E. J., Hernando, C. E., Polcownuk, S., Bertolin, A. P., Mancini, E., Ceriani, M. F. and Yanovsky, M. J. (2017). Rhythmic behavior is controlled by the SRm160 splicing factor in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 28801530
Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kD (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period (per) levels. Moreover, rhythmic accumulation of the neuropeptide Pigment Dispersing Factor (PDF) in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160 downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model.
Bienkowski, R. S., Banerjee, A., Rounds, J. C., Rha, J., Omotade, O. F., Gross, C., Morris, K. J., Leung, S. W., Pak, C., Jones, S. K., Santoro, M. R., Warren, S. T., Zheng, J. Q., Bassell, G. J., Corbett, A. H. and Moberg, K. H. (2017). The conserved, disease-associated RNA binding protein dNab2 interacts with the Fragile X Protein ortholog in Drosophila neurons. Cell Rep 20(6): 1372-1384. PubMed ID: 28793261
The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. This study presents a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.
Yang, L., Titlow, J., Ennis, D., Smith, C., Mitchell, J., Young, F. L., Waddell, S., Ish-Horowicz, D. and Davis, I. (2017). Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains. Methods [Epub ahead of print]. PubMed ID: 28651965
RNA in situ hybridization is a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. This study describes the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary nascent transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.e., exons, 3'UTR, and introns, this paper show examples of a gene that is regulated post-transcriptionally and one that is regulated at the level of transcription. This simple and rapid protocol can be used to co-visualise a variety of different transcripts and proteins in neuronal stem cells as well as deep brain structures such as mushroom body neuropils, using conventional confocal microscopy. Finally, the use of smFISH is introduced as a sensitive alternative to immunofluorescence for labelling specific neural stem cell populations in the brain.
Trcek, T., Lionnet, T., Shroff, H. and Lehmann, R. (2017). mRNA quantification using single-molecule FISH in Drosophila embryos. Nat Protoc 12(7): 1326-1348. PubMed ID: 28594816
Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. This study describes an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e.g., short fluorescently labeled DNA oligonucleotides) in combination with wide-field epifluorescence, confocal or instant structured illumination microscopy (iSIM, a super-resolution imaging approach) and a spot-detection algorithm. Fixed Drosophila embryos are hybridized in solution with a mixture of smFISH probes, mounted onto coverslips and imaged in 3D. Individual fluorescently labeled mRNAs are then localized within tissues and counted using spot-detection software to generate quantitative, spatially resolved gene expression data sets. With minimum guidance, a graduate student can successfully implement this protocol. The smFISH procedure described here can be completed in 4-5 d.
Buchumenski, I., Bartok, O., Ashwal-Fluss, R., Pandey, V., Porath, H. T., Levanon, E. Y. and Kadener, S. (2017). Dynamic hyper-editing underlies temperature adaptation in Drosophila. PLoS Genet 13(7): e1006931. PubMed ID: 28746393
In Drosophila, A-to-I editing is prevalent in the brain, and mutations in the editing enzyme ADAR correlate with specific behavioral defects. This study demonstrates a role for ADAR in behavioral temperature adaptation in Drosophila. Although there is a higher level of editing at lower temperatures, at 29 ° C more sites are edited. These sites are less evolutionarily conserved, more disperse, less likely to be involved in secondary structures, and more likely to be located in exons. Interestingly, hypomorph mutants for ADAR display a weaker transcriptional response to temperature changes than wild-type flies and a highly abnormal behavioral response upon temperature increase. In sum, these data shows that ADAR is essential for proper temperature adaptation, a key behavior trait that is essential for survival of flies in the wild. Moreover, the results suggest a more general role of ADAR in regulating RNA secondary structures in vivo.
Caygill, E. E. and Brand, A. H. (2017). miR-7 buffers differentiation in the developing Drosophila visual system. Cell Rep 20(6): 1255-1261. PubMed ID: 28793250
The 40,000 neurons of the medulla, the largest visual processing center of the Drosophila brain, derive from a sheet of neuroepithelial cells. During larval development, a wave of differentiation sweeps across the neuroepithelium, converting neuroepithelial cells into neuroblasts that sequentially express transcription factors specifying different neuronal cell fates. The switch from neuroepithelial cells to neuroblasts is controlled by a complex gene regulatory network and is marked by the expression of the proneural gene l'sc. This study discovered that microRNA miR-7 is expressed at the transition between neuroepithelial cells and neuroblasts. miR-7 promotes neuroepithelial cell-to-neuroblast transition by targeting downstream Notch effectors to limit Notch signaling. miR-7 acts as a buffer to ensure that a precise and stereotypical pattern of transition is maintained, even under conditions of environmental stress, echoing the role that miR-7 plays in the eye imaginal disc. This common mechanism reflects the importance of robust visual system development.

Friday, August 25th

Pan, J. W., McLaughlin, J., Yang, H., Leo, C., Rambarat, P., Okuwa, S., Monroy-Eklund, A., Clark, S., Jones, C. D. and Volkan, P. C. (2017). Comparative analysis of behavioral and transcriptional variation underlying CO2 sensory neuron function and development in Drosophila. Fly (Austin): [Epub ahead of print]. PubMed ID: 28644712
Carbon dioxide is an important environmental cue for many insects, regulating many behaviors including some that have direct human impacts. To further improve understanding of how this system varies among closely related insect species, both the behavioral response to CO2 as well as the transcriptional profile were examined of key developmental regulators of CO2 sensory neurons in the olfactory system across the Drosophila genus. CO2 was found to generally evoke repulsive behavior across most of the Drosophilids examined, but this behavior has been lost or reduced in several lineages. Comparisons of transcriptional profiles from the developing and adult antennae for subset these species suggest that behavioral differences in some species may be due to differences in the expression of the CO2 co-receptor Gr63a. Furthermore, these differences in Gr63a expression are correlated with changes in the expression of a few genes known to be involved in the development of the CO2 circuit, namely dachshund, an important regulator of sensilla fate for sensilla that house CO2 ORNs, and mip120, a member of the MMB/dREAM epigenetic regulatory complex that regulates CO2 receptor expression. In contrast, most of the other known structural, molecular, and developmental components of the peripheral Drosophila CO2 olfactory system seem to be well-conserved across all examined lineages. These findings suggest that certain components of CO2 sensory ORN development may be more evolutionarily labile, and may contribute to differences in CO2-evoked behavioral responses across species.
Ahmed-Braimah, Y. H., Unckless, R. L. and Clark, A. G. (2017). Evolutionary dynamics of male reproductive genes in the Drosophila virilis subgroup. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 28739599
Postcopulatory sexual selection (PCSS) is a potent evolutionary force that can drive rapid changes of reproductive genes within species, and thus has the potential to generate reproductive incompatibilities between species. Male seminal fluid proteins (SFPs) are major players in postmating interactions, and are important targets of PCSS in males. The virilis subgroup of Drosophila exhibits strong interspecific gametic incompatibilities, and can serve as a model to study the genetic basis of PCSS and gametic isolation. However, reproductive genes in this group have not been characterized. This study utilized short-read RNA sequencing of male reproductive organs to examine the evolutionary dynamics of reproductive genes in members of the virilis subgroup: D. americana, D. lummei, D. novamexicana, and D. virilis. The majority of male reproductive transcripts are testes-biased, accounting for ~15% of all annotated genes. Ejaculatory bulb-biased transcripts largely code for lipid metabolic enzymes, and contain orthologs of the D. melnaogaster ejaculatory bulb protein, Peb-me, which is involved in mating-plug formation. In addition, 71 candidate SFPs were identified, and this gene set was show to have the highest rate of nonsynonymous codon substitution relative to testes- and ejaculatory bulb-biased genes. Furthermore, orthologs were identified of 35 D. melanogaster SFPs that have conserved accessory gland expression in the virilis group. Finally, several of the SFPs that have the highest rate of nonsynonymous codon substitution were shown to reside on chromosomal regions that contribute to paternal gametic incompatibility between species. These results show that SFPs rapidly diversify in the virilis group, and suggest that they likely play a role in PCSS and/or gametic isolation.
Syed, Z. A., Chatterjee, M., Samant, M. A. and Prasad, N. G. (2017). Reproductive isolation through experimental manipulation of sexually antagonistic coevolution in Drosophila melanogaster. Sci Rep 7(1): 3330. PubMed ID: 28611437
Promiscuity can drive the evolution of sexual conflict before and after mating occurs. Post mating, the male ejaculate can selfishly manipulate female physiology, leading to a chemical arms race between the sexes. Theory suggests that drift and sexually antagonistic coevolution can cause allopatric populations to evolve different chemical interactions between the sexes, thereby leading to postmating reproductive barriers and speciation. There is, however, little empirical evidence supporting this form of speciation. This theory was tested by creating an experimental evolutionary model of Drosophila melanogaster populations undergoing different levels of interlocus sexual conflict. Allopatric populations under elevated sexual conflict were shown to exhibit assortative mating, indicating premating reproductive isolation. Further, these allopatric populations also show reduced copulation duration and sperm defense ability when mating happens between individuals across populations compared to that within the same population, indicating postmating prezygotic isolation. Sexual conflict can cause reproductive isolation in allopatric populations through the coevolution of chemical (postmating prezygotic) as well as behavioural (premating) interactions between the sexes. Thus it study provides the first comprehensive evidence of postmating (as well as premating) reproductive isolation due to sexual conflict.
Ribeiro, L., Tobias-Santos, V., Santos, D., Antunes, F., Feltran, G., de Souza Menezes, J., Aravind, L., Venancio, T. M. and Nunes da Fonseca, R. (2017). Evolution and multiple roles of the Pancrustacea specific transcription factor Zelda in insects. PLoS Genet 13(7): e1006868. PubMed ID: 28671979
Gene regulatory networks (GRNs) evolve as a result of the coevolutionary processes acting on transcription factors (TFs) and the cis-regulatory modules they bind. The zinc-finger TF zelda (zld) is essential for the maternal-to-zygotic transition (MZT) in Drosophila melanogaster, where it directly binds over thousand cis-regulatory modules to regulate chromatin accessibility. D. melanogaster displays a long germ type of embryonic development, where all segments are simultaneously generated along the whole egg. However, it remains unclear if zld is also involved in the MZT of short-germ insects (including those from basal lineages) or in other biological processes. This study shows that zld is an innovation of the Pancrustacea lineage, being absent in more distant arthropods (e.g. chelicerates) and other organisms. To better understand the zld ancestral function, its roles were examined in a short-germ beetle, Tribolium castaneum, using molecular biology and computational approaches. The results demonstrate roles for zld not only during the MZT, but also in posterior segmentation and patterning of imaginal disc derived structures. Further, it was also demonstrated that zld is critical for posterior segmentation in the hemipteran Rhodnius prolixus, indicating this function predates the origin of holometabolous insects and was subsequently lost in long-germ insects. These results unveil new roles of zld in different biological contexts and suggest that changes in expression of zld (and probably other major TFs) are critical in the evolution of insect GRNs (Ribeiro, 2017).

Thursday, August 24th

Koltun, B., Shackelford, E., Bonnay, F., Matt, N., Reichhart, J. M. and Orian, A. (2017). The SUMO-targeted ubiquitin ligase, Dgrn, is essential for Drosophila innate immunity. Int J Dev Biol 61(3-4-5): 319-327. PubMed ID: 28621429
This paper reports that Degringolade (Dgrn), a SUMO-targeted ubiquitin ligase connecting the two pathways, is essential for the innate immunity response in Drosophila. dgrnDK null and heterozygous mutant adult flies are severely immune-compromised and succumb rapidly to both pathogenic bacteria and fungi infections. The sensitivity to infection stems from the inability to produce multiple anti-microbial peptides, and transcriptional analyses suggest that the overexpression of Dgrn enhances the transcriptional output of the NF-κB related Toll and immune deficiency (IMD)-pathways. Moreover, expression of Dgrn alleviated the inhibitory impact of the cytoplasmic NF-κB inhibitor Cactus and the nuclear co-repressor Groucho/TLE (Gro). Additionally, Dgrn was found to be required for the local regenerative response of the midgut following infection. Upon oral infection, dgrn mutant flies fail to activate the Delta-Notch pathway in stem cells and enteroblasts, and are unable to regenerate and replace the damaged and dying enterocytes. Interestingly, the ubiquitin-specific protease CG8334 (dUSP32/dUSP11) antagonizes Dgrn activity in the gut, and halving the dose of CG8334 restores Delta-Notch signaling and rescues the lethality observed in dgrn mutants. Collectively, these data suggest that Dgrn is essential for both systemic and local tissue response to infection.
Atilano, M. L., Glittenberg, M., Monteiro, A., Copley, R. and Ligoxygakis, P. (2017). MicroRNAs that contribute to coordinating the immune response in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 28706002
Small noncoding RNAs called microRNAs (miRNAs) have emerged as post-transcriptional regulators of gene expression related to host defences. This study used Drosophila melanogaster to explore the contribution of individual or clusters of miRNAs in countering systemic C. albicans infection. From a total of 72 tested, six miRNAs allelic mutant backgrounds were identified that modulate the survival response to infection and ability to control pathogen number. These mutants also exhibit dysregulation of the Toll pathway target transcripts Drosomycin (Drs) and Immune-Induced Molecule 1 (IM1). These are characteristics of defects in Toll signalling, and consistent with this, dependency for one of the miRNA mutants on the NF-κB homologue Dif was demonstrated. Changes were quantified in the miRNA expression profile over time in response to three pathogen types, and 13 mature miRNA forms affected by pathogens were identified that stimulate Toll signalling. To complement this, a genome-wide map is provided of potential NF-κB sites in proximity to miRNA genes. Finally, systemic C. albicans infection was demonstrated to contribute to a reduction in the total amount of Branch-Chained Amino Acids, which is miRNA-regulated. Overall, these data reveal a new layer of miRNA complexity regulating the fly response to systemic fungal infection.
Aittomaki, S., Valanne, S., Lehtinen, T., Matikainen, S., Nyman, T. A., Ramet, M. and Pesu, M. (2017). Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense. Faseb J. [Epub ahead of print] PubMed ID: 28705811
Invading pathogens provoke robust innate immune responses in Dipteran insects, such as Drosophila melanogaster. In a systemic bacterial infection, a humoral response is induced in the fat body. Gram-positive bacteria trigger the Toll signaling pathway, whereas gram-negative bacterial infections are signaled via the immune deficiency (IMD) pathway. This study shows that the RNA interference-mediated silencing of Furin1-a member of the proprotein convertase enzyme family-specifically in the fat body, results in a reduction in the expression of antimicrobial peptides. This, in turn, compromises the survival of adult fruit flies in systemic infections that are caused by both gram-positive and -negative bacteria. Furin1 plays a nonredundant role in the regulation of immune responses, as silencing of Furin2, the other member of the enzyme family, had no effect on survival or the expression of antimicrobial peptides upon a systemic infection. Furin1 does not directly affect the Toll or IMD signaling pathways, but the reduced expression of Furin1 up-regulates stress response factors in the fat body. This study also demonstrated that Furin1 is a negative regulator of the JAK/STAT signaling pathway, which is implicated in stress responses in the fly. In summary, these data identify Furin1 as a novel regulator of humoral immunity and cellular stress responses in Drosophila.
Zhong, X., Chowdhury, M., Li, C. F. and Yu, X. Q. (2017). Transcription factor Forkhead regulates expression of antimicrobial peptides in the tobacco hornworm, Manduca sexta. Sci Rep 7(1): 2688. PubMed ID: 28578399
Antimicrobial peptides (AMPs) play an important role in defense against microbial infections in insects. Expression of AMPs is regulated mainly by NF-κB factors Dorsal, Dif and Relish. Previous work has shown that both NF-kappaB and GATA-1 factors are required for activation of moricin promoter in the tobacco hornworm, Manduca sexta, and a 140-bp region in the moricin promoter contains binding sites for additional transcription factors. This study, we identified three forkhead (Fkh)-binding sites in the 140-bp region of the moricin promoter and several Fkh-binding sites in the lysozyme promoter, and demonstrated that Fkh-binding sites are required for activation of both moricin and lysozyme promoters by Fkh factors. In addition, Fkh mRNA was undetectable in Drosophila S2 cells, and M. sexta Fkh (MsFkh; see Drosophila Fkh) interacted with Relish-Rel-homology domain (RHD) but not with Dorsal-RHD. Dual luciferase assays with moricin mutant promoters showed that co-expression of MsFkh with Relish-RHD did not have an additive effect on the activity of moricin promoter, suggesting that MsFkh and Relish regulate moricin activation independently. These results suggest that insect AMPs can be activated by Fkh factors under non-infectious conditions, which may be important for protection of insects from microbial infection during molting and metamorphosis.
Wong, C. O., Gregory, S., Hu, H., Chao, Y., Sepulveda, V. E., He, Y., Li-Kroeger, D., Goldman, W. E., Bellen, H. J. and Venkatachalam, K. (2017). Lysosomal degradation is required for sustained phagocytosis of bacteria by macrophages. Cell Host Microbe 21(6): 719-730.e716. PubMed ID: 28579255
Clearance of bacteria by macrophages involves internalization of the microorganisms into phagosomes, which are then delivered to endolysosomes for enzymatic degradation. These spatiotemporally segregated processes are not known to be functionally coupled. This study shows that lysosomal degradation of bacteria sustains phagocytic uptake. In Drosophila and mammalian macrophages, lysosomal dysfunction due to loss of the endolysosomal Cl- transporter ClC-b/CLCN7 delayed degradation of internalized bacteria. Unexpectedly, defective lysosomal degradation of bacteria also attenuated further phagocytosis, resulting in elevated bacterial load. Exogenous application of bacterial peptidoglycans restored phagocytic uptake in the lysosomal degradation-defective mutants via a pathway requiring cytosolic pattern recognition receptors and NF-κB. Mammalian macrophages that are unable to degrade internalized bacteria also exhibit compromised NF-κB activation. These findings reveal a role for phagolysosomal degradation in activating an evolutionarily conserved signaling cascade, which ensures that continuous uptake of bacteria is preceded by lysosomal degradation of microbes.
Shokal, U. and Eleftherianos, I. (2017). The Drosophila Thioester containing Protein-4 participates in the induction of the cellular immune response to the pathogen Photorhabdus. Dev Comp Immunol 76: 200-208. PubMed ID: 28642050
The function of thioester-containing proteins (TEPs) in the immune defense of the fruit fly Drosophila melanogaster is yet mostly unexplored. Recent work has shown the involvement of TEP4 in the activation of humoral and phenoloxidase immune responses of Drosophila against the pathogenic bacteria Photorhabdus luminescens and Photorhabdus asymbiotica. This study investigated the participation of Tep4 in the cellular defense of Drosophila against the two pathogens. Significantly lower numbers of live and dead plasmatocytes are reported in the tep4 mutants compared to control flies in response to Photorhabdus infection. Fewer crystal cells were found in the control flies than in tep4 mutants upon infection with Photorhabdus. These results further suggest that Drosophila hemocytes constitute a major source for the transcript levels of Tep4 in flies infected by Photorhabdus. Finally, Tep4 was shown to participate in the phagocytic function in Drosophila adult flies. Collectively these data support the protective role for TEP4 in the cellular immune response of Drosophila against the entomopathogen Photorhabdus.

Wednesday, August 23rd

Stanley, P. D., Ng'oma, E., O'Day, S. and King, E. G. (2017). Genetic dissection of nutrition-induced plasticity in insulin/insulin-like growth factor signaling and median life span in a Drosophila multiparent population. Genetics 206(2): 587-602. PubMed ID: 28592498
The insulin/insulin-like growth factor signaling (IIS) and target of rapamycin (TOR) pathways have been implicated in nutrition-dependent changes in metabolism and nutrient allocation. To characterize natural genetic variation in the IIS/TOR pathway, >250 recombinant inbred lines (RILs) derived from a multiparental mapping population, the Drosophila Synthetic Population Resource, was used to map transcript-level QTL of genes encoding 52 core IIS/TOR components in three different nutritional environments [dietary restriction (DR), control (C), and high sugar (HS)]. Nearly all genes, 87%, were significantly differentially expressed between diets, though not always in ways predicted by loss-of-function mutants. Cis (i.e., local) expression QTL (eQTL) were identified for six genes, all of which are significant in multiple nutrient environments. Further, trans (i.e., distant) eQTL were identified for two genes, specific to a single nutrient environment. The results are consistent with many small changes in the IIS/TOR pathways. A discriminant function analysis for the C and DR treatments identified a pattern of gene expression associated with the diet treatment. Mapping the composite discriminant function scores revealed a significant global eQTL within the DR diet. A correlation between the discriminant function scores and the median life span (r = 0.46) provides evidence that gene expression changes in response to diet are associated with longevity in these RILs.
Regalado, J. M., Cortez, M. B., Grubbs, J., Link, J. A., van der Linden, A. and Zhang, Y. (2017). Increased food intake after starvation enhances sleep in Drosophila melanogaster. J Genet Genomics 44(6): 319-326. PubMed ID: 28645777
Feeding and sleep are highly conserved, interconnected behaviors essential for survival. Starvation has been shown to potently suppress sleep across species; however, whether satiety promotes sleep is still unclear. This study used the fruit fly, Drosophila melanogaster, as a model organism to address the interaction between feeding and sleep. The sleep of flies that had been starved for 24 h was monitored, and sleep amount was found to increase in the first 4 h after flies were given food. Increased sleep after starvation was due to an increase in sleep bout number and average sleep bout length. Mutants of translin or adipokinetic hormone, which fail to suppress sleep during starvation, still exhibited a sleep increase after starvation, suggesting that sleep increase after starvation is not a consequence of sleep loss during starvation. It was also found that feeding activity and food consumption were higher in the first 10-30 min after starvation. Restricting food consumption in starved flies to 30 min was sufficient to increase sleep for 1 h. Although flies ingested a comparable amount of food at differing sucrose concentrations, sleep increase after starvation on a lower sucrose concentration was undetectable. Taken together, these results suggest that increased food intake after starvation enhances sleep and reveals a novel relationship between feeding and sleep.
Polesello, C. and Le Bourg, E. (2017). A mild cold stress that increases resistance to heat lowers FOXO translocation in Drosophila melanogaster. Biogerontology [Epub ahead of print]. PubMed ID: 28677014
Previous studies have shown that subjecting Drosophila melanogaster flies to a mild stress at young or middle age can increase lifespan and resistance to severe stresses throughout life and that the NF-kappaB-like transcription factor DIF, the 70 kDa heat-shock proteins, and the Drosophila Forkhead box class O (dFOXO) transcription factor could explain some of these effects. The present study showed that two dFOXO mutants do not survive longer heat if previously subjected to a mild cold stress, contrarily to wild-type flies. This cold pretreatment had nearly no effect on dFOXO nuclear translocation in wild-type males. Heat stress strongly increased dFOXO translocation, but this effect was lowered in cold-pretreated males. Because cold-pretreated wild-type males survived longer heat and had nevertheless a lower dFOXO translocation after this heat stress, one can conclude that dFOXO is required to resist heat but that the cold pretreatment makes that other mechanisms partly substitute to dFOXO translocation.
Zandveld, J., van den Heuvel, J., Zwaan, B. J. and Piper, M. D. W. (2017). Both overlapping and independent mechanisms determine how diet and insulin-ligand knockouts extend lifespan of Drosophila melanogaster. NPJ Aging Mech Dis 3: 4. PubMed ID: 28649422
Lifespan in many organisms, including Drosophila melanogaster, can be increased by reduced insulin-IGF-like signaling (IIS) or by changes in diet. Most studies testing whether IIS is involved in diet-mediated lifespan extension employ only a few diets, but recent data shows that a broad range of nutritional environments is required. This study presents lifespan data of long-lived Drosophila, lacking three of the eight insulin-like peptides [Drosophila insulin-like peptides 2,3,5 (dilp2-3,5)] on nine different diets that surround the optimum for lifespan. Their nutritional content was varied by manipulating sugar and yeast concentrations independently, and thus incorporated changes in both diet restriction and nutrient balance. The mutants were substantially longer-lived than controls on every diet, but the effects on the lifespan response to sugar and yeast differed. The data illustrates how a greater coverage of diet balance (DB) and restriction can unify differing interpretations of how IIS might be involved in the response of lifespan to diet.

Tuesday, August 22nd

Panchal, T., Chen, X., Alchits, E., Oh, Y., Poon, J., Kouptsova, J., Laski, F. A. and Godt, D. (2017). Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam. PLoS Genet 13(5): e1006790. PubMed ID: 28542174
Germline stem cells in the Drosophila ovary are maintained by a somatic niche. The niche is structurally and functionally complex and contains four cell types, the escort, cap, and terminal filament cells and the newly identified transition cell. The large Maf transcription factor Traffic jam (Tj) is essential for determining niche cell fates and architecture, enabling each niche in the ovary to support a normal complement of 2-3 germline stem cells. In particular, this study focused on the question of how cap cells form. Cap cells express Tj and are considered the key component of a mature germline stem cell niche. It is concluded that Tj controls the specification of cap cells, as the complete loss of Tj function caused the development of additional terminal filament cells at the expense of cap cells, and terminal filament cells developed cap cell characteristics when induced to express Tj. The data indicate that Tj contributes to the establishment of germline stem cells by promoting the cap cell fate, and controls the stem cell-carrying capacity of the niche by regulating niche architecture. Analysis of the interactions between Tj and the Notch (N) pathway indicates that Tj and N have distinct functions in the cap cell specification program. It is proposed that formation of cap cells depends on the combined activities of Tj and the N pathway, with Tj promoting the cap cell fate by blocking the terminal filament cell fate, and N supporting cap cells by preventing the escort cell fate and/or controlling the number of cap cell precursors.
Koehler, C. L., Perkins, G. A., Ellisman, M. H. and Jones, D. L. (2017). Pink1 and Parkin regulate Drosophila intestinal stem cell proliferation during stress and aging. J Cell Biol. PubMed ID: 28663346
Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. This study investigated how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. These results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles.
Wu, Y. C., Lee, K. S., Song, Y., Gehrke, S. and Lu, B. (2017). The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain. PLoS Genet 13(5): e1006785. PubMed ID: 28520736
Notch (N) signaling is central to the self-renewal of neural stem cells (NSCs) and other tissue stem cells. Its deregulation compromises tissue homeostasis and contributes to tumorigenesis and other diseases. How N regulates stem cell behavior in health and disease is not well understood. This study shows that Notch regulates bantam (ban) microRNA to impact cell growth, a process key to NSC maintenance and particularly relied upon by tumor-forming cancer stem cells. Notch signaling directly regulates ban expression at the transcriptional level, and ban in turn feedback regulates N activity through negative regulation of the Notch inhibitor Numb. This feedback regulatory mechanism helps maintain the robustness of N signaling activity and NSC fate. Moreover, this study shows that a Numb-Myc axis mediates the effects of ban on nucleolar and cellular growth independently or downstream of N. These results highlight intricate transcriptional as well as translational control mechanisms and feedback regulation in the N signaling network, with important implications for NSC biology and cancer biology.
Xu, C., Luo, J., He, L., Montell, C. and Perrimon, N. (2017). Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut. Elife 6. PubMed ID: 28561738
Precise regulation of stem cell activity is crucial for tissue homeostasis and necessary to prevent overproliferation. In the Drosophila adult gut, high levels of reactive oxygen species (ROS) has been detected with different types of tissue damage, and oxidative stress has been shown to be both necessary and sufficient to trigger intestinal stem cell (ISC) proliferation. However, the connection between oxidative stress and mitogenic signals remains obscure. In a screen for genes required for ISC proliferation in response to oxidative stress, this study identified two regulators of cytosolic Ca2+ levels, transient receptor potential A1 (TRPA1) and ryanodine receptor (RyR). Characterization of TRPA1 and RyR demonstrates that Ca2+ signaling is required for oxidative stress-induced activation of the Ras/MAPK pathway, which in turns drives ISC proliferation. These findings provide a link between redox regulation and Ca2+ signaling and reveal a novel mechanism by which ISCs detect stress signals.

Monday, August 21st

Wortman, J. C., Nahmad, M., Zhang, P. C., Lander, A. D. and Yu, C. C. (2017). Expanding signaling-molecule wavefront model of cell polarization in the Drosophila wing primordium. PLoS Comput Biol 13(7): e1005610. PubMed ID: 28671940
Cells throughout the wing primordium typically show subcellular localization of the unconventional myosin Dachs on the distal side of cells (nearest the center of the disc). Dachs localization depends on the spatial distribution of bonds between the protocadherins Fat (Ft) and Dachsous (Ds), which form heterodimers between adjacent cells; and the Golgi kinase Four-jointed (Fj), which affects the binding affinities of Ft and Ds. The Fj concentration forms a linear gradient while the Ds concentration is roughly uniform throughout most of the wing pouch with a steep transition region that propagates from the center to the edge of the pouch during the third larval instar. It is unclear how the polarization is affected by cell division and the expanding Ds transition region, both of which can alter the distribution of Ft-Ds heterodimers around the cell periphery. A computational model was developed to address these questions. In this model, the binding affinity of Ft and Ds depends on phosphorylation by Fj. It is assumed that the asymmetry of the Ft-Ds bond distribution around the cell periphery defines the polarization, with greater asymmetry promoting cell proliferation. The model predicts that this asymmetry is greatest in the radially-expanding transition region that leaves polarized cells in its wake. These cells naturally retain their bond distribution asymmetry after division by rapidly replenishing Ft-Ds bonds at new cell-cell interfaces. Thus it is predicted that the distal localization of Dachs in cells throughout the pouch requires the movement of the Ds transition region and the simple presence, rather than any specific spatial pattern, of Fj.
Kumar, A. and Tiwari, A. K. (2017). Molecular chaperone Hsp70 and its constitutively active form Hsc70 play an indispensable role during eye development of Drosophila melanogaster. Mol Neurobiol. PubMed ID: 28634860
This study demonstrates that molecular chaperone Hsp70 and Hsc70 is essential for normal organization and development of ommatidial cells in Drosophila melanogaster eye. A dominant negative mutant of Hsp70 (K71E) and Hsc70.4 (K71S and D206S) exogenously expressed in an eye-specific manner resulted in eye degeneration that includes loss of eye pigment, disorganized ommatidia, abnormality in bristle cell arrangement and reduction in the eye size. The developmental organization of ommatidial cells (cone, photoreceptor, pigment, and bristle cell complex) was disturbed in Hsp70 and Hsc70 mutants. Acridine orange (AO) and caspase 3 staining showed an increased cell death in Hsp70 and Hsc70 mutant eyes. Genetic interaction study of Hsp70 and Hsc70 mutants with candidate genes of JNK signaling pathway and immunocytochemistry study using phospho-JNK antibody suggested that mutation in Hsp70 and Hsc70 results in ectopic activation of JNK signaling in fly eye. Further, anti-PH3 staining in Hsp70 and Hsc70 mutant eyes revealed a reduced number of mitotic cells in second mitotic wave (SMW) of developing eye and anti-Rh1 staining showed reduced Rh1 expression, accumulation of Rh1 in the cytoplasm, and rhabdomere degeneration. Thus, on the basis of results, it was concluded that molecular chaperone Hsp70 and Hsc70 play an indispensable role during Drosophila eye development.
Meserve, J. H. and Duronio, R. J. (2017). A population of G2-arrested cells are selected as sensory organ precursors for the interommatidial bristles of the Drosophila eye. Dev Biol [Epub ahead of print]. PubMed ID: 28645749
Cell cycle progression and differentiation are highly coordinated during the development of multicellular organisms. The mechanisms by which these processes are coordinated and how their coordination contributes to normal development are not fully understood. This study determined the developmental fate of a population of precursor cells in the developing Drosophila melanogaster retina that arrest in G2 phase of the cell cycle and investigated whether cell cycle phase-specific arrest influences the fate of these cells. Retinal precursor cells that arrest in G2 during larval development were shown to be selected as sensory organ precursors (SOPs) during pupal development and undergo two cell divisions to generate the four-cell interommatidial mechanosensory bristles. While G2 arrest is not required for bristle development, preventing G2 arrest results in incorrect bristle positioning in the adult eye. It is concluded that G2-arrested cells provide a positional cue during development to ensure proper spacing of bristles in the eye. The results suggest that the control of cell cycle progression refines cell fate decisions and that the relationship between these two processes is not necessarily deterministic.
Matsuda, S. and Affolter, M. (2017). Dpp from the anterior stripe of cells is crucial for the growth of the Drosophila wing disc. Elife 6. PubMed ID: 28675373
The Dpp morphogen gradient derived from the anterior stripe of cells is thought to control growth and patterning of the Drosophila wing disc. However, the spatial-temporal requirement of dpp for growth and patterning remained largely unknown. Recently, two studies re-addressed this question. By generating a conditional null allele, one study proposed that the dpp stripe is critical for patterning but not for growth. In contrast, using a membrane-anchored nanobody to trap Dpp, the other study proposed that Dpp dispersal from the stripe is required for patterning and also for medial wing disc growth, at least in the posterior compartment. Thus, growth control by the Dpp morphogen gradient remains under debate. By removing dpp from the stripe at different time points, this study shows that the dpp stripe source is indeed required for wing disc growth, also during third instar larval stages.

Sunday, August 20th

Revaitis, N. T., Marmion, R. A., Farhat, M., Ekiz, V., Wang, W. and Yakoby, N. (2017). Simple expression domains are regulated by discrete CRMs during Drosophila oogenesis. G3 (Bethesda). PubMed ID: 28634244
Eggshell patterning has been extensively studied in Drosophila melanogaster. However, the cis-regulatory modules (CRMs), which control spatiotemporal expression of these patterns, are vastly unexplored. The FlyLight collection contains over 7,000 intergenic and intronic DNA fragments that, if containing CRMs, can drive the transcription factor GAL4. The 84 genes known to be expressed during D. melanogaster oogenesis were cross-listed with the ~1200 listed genes of the FlyLight collection, and 22 common genes were found that are represented by 281 FlyLight fly lines. Of these lines, 54 show expression patterns during oogenesis when crossed to an UAS-GFP reporter. Of the 54 lines, 16 recapitulate the full or partial pattern of the associated gene pattern. Interestingly, while the average DNA fragment size is ~3kb in length, the vast majority of fragments show one type of a spatiotemporal pattern in oogenesis. Mapping the distribution of all 54 lines, a significant enrichment of CRMs was found in the first intron of the associated genes' model. In addition, the use was demonstrated of different anteriorly active FlyLight lines as tools to disrupt eggshell patterning in a targeted manner. This screen provides further evidence that complex gene-patterns are assembled combinatorially by different CRMs controlling the expression of genes in simple domains.
Zhang, C., Montooth, K. L. and Calvi, B. R. (2017). Incompatibility between mitochondrial and nuclear genomes during oogenesis results in ovarian failure and embryonic lethality. Development 144(13): 2490-2503. PubMed ID: 28576772
Mitochondrial dysfunction can cause female infertility. An important unresolved issue is the extent to which incompatibility between mitochondrial and nuclear genomes contributes to female infertility. It has previously been shown that a mitochondrial haplotype from D. simulans (simw501) is incompatible with a nuclear genome from the D. melanogaster strain Oregon-R (OreR), resulting in impaired development, which was enhanced at higher temperature. This mito-nuclear incompatibility is between alleles of the nuclear-encoded mitochondrial tyrosyl-tRNA synthetase (Aatm) and the mitochondrial-encoded tyrosyl-tRNA that it aminoacylates. This study shows that this mito-nuclear incompatibility causes a severe temperature-sensitive female infertility. The OreR nuclear genome contributed to death of ovarian germline stem cells and reduced egg production, which was further enhanced by the incompatibility with simw501 mitochondria. Mito-nuclear incompatibility also resulted in aberrant egg morphology and a maternal-effect on embryonic chromosome segregation and survival, which was completely dependent on the temperature and mito-nuclear genotype of the mother. These findings show that maternal mito-nuclear incompatibility during Drosophila oogenesis has severe consequences for egg production and embryonic survival, with important broader relevance to human female infertility and mitochondrial replacement therapy.
Manning, L., Sheth, J., Bridges, S., Saadin, A., Odinammadu, K., Andrew, D., Spencer, S., Montell, D. and Starz-Gaiano, M. (2017). A hormonal cue promotes timely follicle cell migration by modulating transcription profiles. Mech Dev [Epub ahead of print]. PubMed ID: 28610887
Cell migration is essential during animal development. In the Drosophila ovary, the steroid hormone ecdysone coordinates nutrient sensing, growth, and the timing of morphogenesis events including border cell migration. To identify downstream effectors of ecdysone signaling, this study profiled gene expression in wild-type follicle cells compared to cells expressing a dominant negative Ecdysone receptor or its coactivator Taiman. Of approximately 400 genes that showed differences in expression, 16 candidate genes were validated for expression in border and centripetal cells, and seven responded to ectopic ecdysone activation by changing their transcriptional levels. A requirement was found for seven putative targets in effective cell migration, including two other nuclear hormone receptors, a calcyphosine-encoding gene, and a prolyl hydroxylase. Thus, this study identified multiple new genetic regulators modulated at the level of transcription that allow cells to interpret information from the environment and coordinate cell migration in vivo.
Tan, S. W. S., Lee, Q. Y., Wong, B. S. E., Cai, Y. and Baeg, G. H. (2017). Redox homeostasis plays important roles in the maintenance of the Drosophila testis germline stem cells. Stem Cell Reports [Epub ahead of print]. PubMed ID: 28669604
Oxidative stress influences stem cell behavior by promoting the differentiation, proliferation, or apoptosis of stem cells. Thus, characterizing the effects of reactive oxygen species (ROS) on stem cell behavior provides insights into the significance of redox homeostasis in stem cell-associated diseases and efficient stem cell expansion for cellular therapies. This study utilized the Drosophila testis as an in vivo model to examine the effects of ROS on germline stem cell (GSC) maintenance. High levels of ROS induced by alteration in activity of Nrf2 and its cytoplasmic inhibitor Keap1 decreased GSC number by promoting precocious GSC differentiation. Notably, high ROS enhanced the transcription of the EGFR ligand spitz and the expression of phospho-Erk1/2, suggesting that high ROS-mediated GSC differentiation is through EGFR signaling. By contrast, testes with low ROS caused by Keap1 inhibition or antioxidant treatment showed an overgrowth of GSC-like cells. These findings suggest that redox homeostasis regulated by Keap1/Nrf2 signaling plays important roles in GSC maintenance.

Saturday, August 19th

Chen, J. V., Buchwalter, R. A., Kao, L. R. and Megraw, T. L. (2017). A splice variant of Centrosomin converts mitochondria to microtubule-organizing centers. Curr Biol 27(13): 1928-1940 e1926. PubMed ID: 28669756
Non-centrosomal microtubule organizing centers (MTOCs) direct microtubule (MT) organization to exert diverse cell-type-specific functions. In Drosophila spermatids, the giant mitochondria provide structural platforms for MT reorganization to support elongation of the extremely long sperm. However, the molecular basis for this mitochondrial MTOC and other non-centrosomal MTOCs has not been discerned. This study reports that Drosophila centrosomin (cnn) expresses two major protein variants: the centrosomal form (CnnC) and a non-centrosomal form in testes (CnnT). CnnC is established as essential for functional centrosomes, the major MTOCs in animal cells. This study shows that CnnT is expressed exclusively in testes by alternative splicing and localizes to giant mitochondria in spermatids. In cell culture, CnnT targets to the mitochondrial surface, recruits the MT nucleator γ-tubulin ring complex (gamma-TuRC), and is sufficient to convert mitochondria to MTOCs independent of core pericentriolar proteins that regulate MT assembly at centrosomes. Two separate domains in CnnT were mapped: one that is necessary and sufficient to target it to mitochondria and another that is necessary and sufficient to recruit gamma-TuRCs and nucleate MTs. In elongating spermatids, CnnT forms speckles on the giant mitochondria that are required to recruit gamma-TuRCs to organize MTs and support spermiogenesis. This molecular characterization of the mitochondrial MTOC defines a minimal molecular requirement for MTOC generation and implicates the potent role of Cnn (or its related) proteins in the direct regulation of MT assembly and organization of non-centrosomal MTOCs.
Vig, A. T., Foldi, I., Szikora, S., Migh, E., Gombos, R., Toth, M. A., Huber, T., Pinter, R., Talian, G. C., Mihaly, J. and Bugyi, B. (2017). The activities of the c-terminal regions of the formin protein Disheveled-associated activator of morphogenesis (Daam) in actin dynamics. J Biol Chem [Epub ahead of print]. PubMed ID: 28642367
Disheveled-associated activator of morphogenesis (DAAM) is a diaphanous-related formin protein essential for the regulation of actin cytoskeleton dynamics in diverse biological processes. The conserved formin homology 1 and 2 (FH1-FH2) domains of DAAM catalyze actin nucleation and processively mediate filament elongation. These activities are indirectly regulated by the N-, and C-terminal regions flanking the FH1-FH2 domains. Recently, the C-terminal diaphanous-autoregulatory domain (DAD) and the C-terminus (CT) of formins have also been shown to regulate actin assembly by directly interacting with actin. To better understand the biological activities of DAAM, the role of DAD-CT regions of Drosophila DAAM in its interaction with actin was studied with in vitro biochemical and in vivo genetic approaches. The DAD-CT region was found to bind actin in vitro and that its main actin-binding element is the CT region, which does not influence actin dynamics on its own. However, it was also found that it can tune the nucleating activity and the filament end-interaction properties of DAAM in an FH2 domain-dependent manner. It was also demonstrated that DAD-CT makes the FH2 domain more efficient in antagonizing with capping protein. Consistently, in vivo data suggested that the CT region contributes to DAAM-mediated filopodia formation and dynamics in primary neurons. In conclusion, these results demonstrate that the CT region of DAAM plays an important role in actin assembly regulation in a biological context.
Tissot, N., Lepesant, J. A., Bernard, F., Legent, K., Bosveld, F., Martin, C., Faklaris, O., Bellaiche, Y., Coppey, M. and Guichet, A. (2017). Distinct molecular cues ensure a robust microtubule-dependent nuclear positioning in the Drosophila oocyte. Nat Commun 8: 15168. PubMed ID: 28447612
Controlling nucleus localization is crucial for a variety of cellular functions. In the Drosophila oocyte, nuclear asymmetric positioning is essential for the reorganization of the microtubule (MT) network that controls the polarized transport of axis determinants. A combination of quantitative three-dimensional live imaging and laser ablation-mediated force analysis reveal that nuclear positioning is ensured with an unexpected level of robustness. The nucleus is pushed to the oocyte antero-dorsal cortex by MTs and that its migration can proceed through distinct tracks. Centrosome-associated MTs favour one migratory route. In addition, the MT-associated protein Mud/NuMA that is asymmetrically localized in an Asp-dependent manner at the nuclear envelope hemisphere where MT nucleation is higher promotes a separate route. These results demonstrate that centrosomes do not provide an obligatory driving force for nuclear movement, but together with Mud, contribute to the mechanisms that ensure the robustness of asymmetric nuclear positioning.
Szikora, S., Foldi, I., Toth, K., Migh, E., Vig, A., Bugyi, B., Maleth, J., Hegyi, P., Kaltenecker, P., Sanchez-Soriano, N. and Mihaly, J. (2017). The formin DAAM is required for coordination of the actin and microtubule cytoskeleton in axonal growth cones. J Cell Sci [Epub ahead of print]. PubMed ID: 28606990
Directed axonal growth depends on proper coordination of the actin and microtubule cytoskeleton in the growth cone. However, despite the relatively large number of proteins implicated in actin-microtubule cross-talk, the mechanisms whereby actin polymerization is coupled to microtubule stabilization and advancement in the peripheral growth cone remained largely unclear. This study identified the formin DAAM as a novel factor playing a role in concerted regulation of actin and microtubule remodeling in Drosophila primary neurons. In vitro DAAM binds to F-actin as well as microtubules and it has the ability to crosslink the two filament systems. Accordingly, DAAM associates with the neuronal cytoskeleton, and a significant fraction of DAAM accumulates at places where the actin filaments overlap with that of microtubules. Loss of DAAM affects growth cone and microtubule morphology and several aspects of microtubule dynamics, whereas biochemical and cellular assays revealed a microtubule stabilization activity and binding to the microtubule tip protein EB1. Together these data suggest that besides operating as an actin assembly factor, DAAM is involved in linking filopodial actin remodeling to microtubule stabilization during axonal growth.
Wioland, H., Guichard, B., Senju, Y., Myram, S., Lappalainen, P., Jegou, A. and Romet-Lemonne, G. (2017). ADF/Cofilin accelerates actin dynamics by severing filaments and promoting their depolymerization at both ends. Curr Biol 27(13): 1956-1967 e1957. PubMed ID: 28625781
Evolutionary Homolog Study
Actin-depolymerizing factor (ADF)/cofilins (see Drosophila Twinstar) contribute to cytoskeletal dynamics by promoting rapid actin filament disassembly. In the classical view, ADF/cofilin sever filaments, and capping proteins (see Drosophila Capulet) block filament barbed ends whereas pointed ends depolymerize, at a rate that is still debated. By monitoring the activity of the three mammalian ADF/cofilin isoforms on individual skeletal muscle and cytoplasmic actin filaments, this study directly quantify the reactions underpinning filament severing and depolymerization from both ends. In the absence of monomeric actin, soluble ADF/cofilin was found to associate with bare filament barbed ends to accelerate their depolymerization. Compared to bare filaments, ADF/cofilin-saturated filaments depolymerize faster from their pointed ends and slower from their barbed ends, resulting in similar depolymerization rates at both ends. This effect is isoform specific because depolymerization is faster for ADF- than for cofilin-saturated filaments. It was also shown that, unexpectedly, ADF/cofilin-saturated filaments qualitatively differ from bare filaments: their barbed ends are very difficult to cap or elongate, and consequently undergo depolymerization even in the presence of capping protein and actin monomers. Such depolymerizing ADF/cofilin-decorated barbed ends are produced during 17% of severing events. They are also the dominant fate of filament barbed ends in the presence of capping protein, because capping allows growing ADF/cofilin domains to reach the barbed ends, thereby promoting their uncapping and subsequent depolymerization. These experiments thus reveal how ADF/cofilin, together with capping protein, control the dynamics of actin filament barbed and pointed ends. Strikingly, the results propose that significant barbed-end depolymerization may take place in cells.
Shekhar, S. and Carlier, M. F. (2017). Enhanced depolymerization of actin filaments by ADF/Cofilin and monomer funneling by capping protein cooperate to accelerate barbed-end growth. Curr Biol 27(13): 1990-1998 e1995. PubMed ID: 28625780
Evolutionary Homolog Study
A living cell's ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers, which feed polarized filament growth. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like actin depolymerizing factor (ADF)/cofilin (see Drosophila Twinstar) and filament cappers like capping protein (CP; see Drosophila Capulet) are essential agonists of motility, but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades. Whereas filament fragmentation by ADF/cofilin has long been demonstrated by total internal reflection fluorescence (TIRF), filament depolymerization was only inferred from bulk solution assays. Using microfluidics-assisted TIRF microscopy, this study provides the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. Using a conceptually novel assay to directly visualize ADF's effect on a population of pre-assembled filaments, it was demonstrated how ADF's enhanced pointed-end depolymerization causes an increase in polymerizable actin monomers, thus promoting faster barbed-end growth. It was further reveale that ADF-enhanced depolymerization synergizes with CP's long-predicted "monomer funneling" and leads to skyrocketing of filament growth rates, close to estimated lamellipodial rates. The "funneling model" hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few non-capped filaments transiently grow much faster, an effect proposed to be very important for motility. This study provides the first direct microscopic evidence of monomer funneling at the scale of individual filaments. These results significantly enhance understanding of the turnover of cellular actin networks.

Friday, August 18th

Prakash, P., Nambiar, A. and Sheeba, V. (2017). Oscillating PDF in termini of circadian pacemaker neurons and synchronous molecular clocks in downstream neurons are not sufficient for sustenance of activity rhythms in constant darkness. PLoS One 12(5): e0175073. PubMed ID: 28558035
In Drosophila, neuropeptide Pigment Dispersing Factor (PDF) is expressed in small and large ventral Lateral Neurons (sLNv and lLNv), among which sLNv are critical for activity rhythms in constant darkness. Studies show that this is mediated by rhythmic accumulation and likely secretion of PDF from sLNv dorsal projections, which in turn synchronises molecular oscillations in downstream circadian neurons. Using targeted expression of a neurodegenerative protein Huntingtin in LNv, a selective loss is evoked of neuropeptide PDF and clock protein Period from sLNv soma. However, PDF is not lost from sLNv dorsal projections and lLNv. These flies are behaviourally arrhythmic in constant darkness despite persistence of PDF oscillations in sLNv dorsal projections and synchronous Period oscillations in downstream circadian neurons. PDF oscillations in sLNv dorsal projections are not sufficient for sustenance of activity rhythms in constant darkness and this is suggestive of an additional component that is possibly dependent on sLNv molecular clock and PDF in sLNv soma. Additionally, despite loss of Period in sLNv, their activity rhythms entrain to light/dark cycles indicating that sLNv molecular clocks are not necessary for entrainment. Under constant light, these flies lack PDF from both soma and dorsal projections of sLNv, and when subjected to light/dark cycles, show morning and evening anticipation and accurately phased morning and evening peaks. Thus, under light/dark cycles, PDF in sLNv is not necessary for morning anticipation.
Selvan, N., Williamson, R., Mariappa, D., Campbell, D. G., Gourlay, R., Ferenbach, A. T., Aristotelous, T., Hopkins-Navratilova, I., Trost, M. and van Aalten, D. M. F. (2017). A mutant O-GlcNAcase enriches Drosophila developmental regulators. Nat Chem Biol. PubMed ID: 28604694
Protein O-GlcNAcylation is a reversible post-translational modification of serines and threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis. The Drosophila melanogaster gene supersex combs (sxc), which encodes OGT, is a polycomb gene, whose null mutants display homeotic transformations and die at the pharate adult stage. However, the identities of the O-GlcNAcylated proteins involved and the underlying mechanisms linking these phenotypes to embryonic development are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is hampered by the low stoichiometry of this modification and by limited enrichment tools. Using a catalytically inactive bacterial O-GlcNAcase mutant as a substrate trap, this study has enriched the O-GlcNAc proteome of the developing Drosophila embryo, identifying, among others, known regulators of Hox genes as candidate conveyors of OGT function during embryonic development.
Sanchez Bosch, P., Ziukaite, R., Alexandre, C., Basler, K. and Vincent, J. B. (2017). Dpp controls growth and patterning in Drosophila wing precursors through distinct modes of action. Elife 6 [Epub ahead of print]. PubMed ID: 28675374
Dpp, a member of the BMP family, is a morphogen that specifies positional information in Drosophila wing precursors. In this tissue, Dpp expressed along the anterior-posterior boundary forms a concentration gradient that controls the expression domains of target genes, which in turn specify the position of wing veins. Dpp also promotes growth in this tissue. The relationship between the spatio-temporal profile of Dpp signalling and growth has been the subject of debate, which has intensified recently with the suggestion that the stripe of Dpp is dispensable for growth. With two independent conditional alleles of dpp this study found that the stripe of Dpp is essential for wing growth. It was then shown that this requirement, but not patterning, can be fulfilled by uniform, low level, Dpp expression. Thus, the stripe of Dpp ensures that signalling remains above a pro-growth threshold, while at the same time generating a gradient that patterns cell fates.
Suisse, A., He, D., Legent, K. and Treisman, J. E. (2017). COP9 signalosome subunits protect Capicua from MAP kinase-dependent and independent mechanisms of degradation. Development [Epub ahead of print]. PubMed ID: 28619822
The COP9 signalosome removes Nedd8 modifications from the Cullin subunits of ubiquitin ligase complexes, reducing their activity. This study shows that mutations in the Drosophila COP9 signalosome subunit 1b (CSN1b) gene increase the activity of ubiquitin ligases that contain Cullin 1. Analysis of CSN1b mutant phenotypes revealed a requirement for the COP9 signalosome to prevent ectopic expression of Epidermal growth factor receptor (EGFR) target genes. It does so by protecting Capicua, a transcriptional repressor of EGFR target genes, from EGFR pathway-dependent ubiquitination by a Cullin 1/SKP1-related A/Archipelago E3 ligase and subsequent proteasomal degradation. The CSN1b subunit also maintains basal Capicua levels by protecting it from a separate mechanism of degradation that is independent of EGFR signaling. As a suppressor of tumor growth and metastasis, Capicua may be an important target of the COP9 signalosome in cancer.

Thursday, August 17th

Shilts, J. and Broadie, K. (2017). Secreted tissue inhibitor of matrix metalloproteinase restricts trans-synaptic signaling to coordinate synaptogenesis. J Cell Sci [Epub ahead of print]. PubMed ID: 28576972
Synaptogenesis is coordinated by trans-synaptic signals that traverse the specialized synaptomatrix between pre- and postsynaptic cells. Matrix metalloproteinase (Mmp) activity sculpts this environment, balanced by secreted Tissue inhibitors of Mmp (Timp). This study used the reductionist Drosophila matrix metalloproteome to test consequences of eliminating all Timp regulatory control of Mmp activity at the neuromuscular junction (NMJ). Using in situ zymography, Timp was found to limit Mmp activity at the NMJ terminal and shape extracellular proteolytic dynamics surrounding individual synaptic boutons. In newly-generated timp null mutants, NMJs exhibit architectural overelaboration with supernumerary synaptic boutons. With cell-targeted RNAi and rescue studies, postsynaptic Timp was found to limit presynaptic architecture. Functionally, timp nulls exhibit compromised synaptic vesicle cycling, with reduced, lower fidelity activity. NMJ defects manifest in impaired locomotor function. Mechanistically, Timp was found to limit BMP trans-synaptic signaling and the downstream synapse-to-nucleus signal transduction. Pharmacologically restoring Mmp inhibition in timp nulls corrects BMP signaling and synaptic properties. Genetically restoring BMP signaling in timp nulls corrects NMJ structure and motor function. Thus, Timp inhibition of Mmp proteolytic activity restricts BMP trans-synaptic signaling to coordinate synaptogenesis.
Wagner, N. (2017). Ultrastructural comparison of the Drosophila larval and adult ventral abdominal neuromuscular junction. J Morphol [Epub ahead of print]. PubMed ID: 28444917
Drosophila melanogaster has recently emerged as model system for studying synaptic transmission and plasticity during adulthood, aging and neurodegeneration. However, still little is known about the basic neuronal mechanisms of synaptic function in the adult fly. Per se, adult Drosophila neuromuscular junctions should be highly suited for studying these aspects as they allow for genetic manipulations in combination with ultrastructural and electrophysiological analyses. Although different neuromuscular junctions of the adult fly have been described during the last years, a direct ultrastructural comparison with their larval counterpart is lacking. The present study was designed to close this gap by providing a detailed ultrastructural comparison of the larval and the adult neuromuscular junction of the ventrolongitudinal muscle. Assessment of several parameters revealed similarities but also major differences in the ultrastructural organisation of the two model neuromuscular junctions. While basic morphological parameters are retained from the larval into the adult stage, the analysis discovered major differences of potential functional relevance in the adult: The electron-dense membrane apposition of the presynaptic and postsynaptic membrane is shorter, the subsynaptic reticulum is less elaborated and the number of synaptic vesicles at a certain distance of the presynaptic membrane is higher.
Kiragasi, B., Wondolowski, J., Li, Y. and Dickman, D. K. (2017). A presynaptic glutamate receptor subunit confers robustness to neurotransmission and homeostatic potentiation. Cell Rep 19(13): 2694-2706. PubMed ID: 28658618
Homeostatic signaling systems are thought to interface with other forms of plasticity to ensure flexible yet stable levels of neurotransmission. The role of neurotransmitter receptors in this process, beyond mediating neurotransmission itself, is not known. Through a forward genetic screen, this study has identified the Drosophila kainate-type ionotropic glutamate receptor subunit DKaiR1D to be required for the retrograde, homeostatic potentiation of synaptic strength. DKaiR1D is necessary in presynaptic motor neurons, localized near active zones, and confers robustness to the calcium sensitivity of baseline synaptic transmission. Acute pharmacological blockade of DKaiR1D disrupts homeostatic plasticity, indicating that this receptor is required for the expression of this process, distinct from developmental roles. Finally, this study demonstrated that calcium permeability through DKaiR1D is necessary for baseline synaptic transmission, but not for homeostatic signaling. It is proposed that DKaiR1D is a glutamate autoreceptor that promotes robustness to synaptic strength and plasticity with active zone specificity.
Mosca, T. J., Luginbuhl, D. J., Wang, I. E. and Luo, L. (2017). Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons. Elife 6. PubMed ID: 28606304
Precise coordination of synaptic connections ensures proper information flow within circuits. The activity of presynaptic organizing molecules signaling to downstream pathways is essential for such coordination, though such entities remain incompletely known. This study shows that LRP4 (CG8909), a conserved transmembrane protein known for its postsynaptic roles, functions presynaptically as an organizing molecule. In the Drosophila brain, LRP4 localizes to the nerve terminals at or near active zones. Loss of presynaptic LRP4 reduces excitatory (not inhibitory) synapse number, impairs active zone architecture, and abolishes olfactory attraction - the latter of which can be suppressed by reducing presynaptic GABAB receptors. LRP4 overexpression increases synapse number in excitatory and inhibitory neurons, suggesting an instructive role and a common downstream synapse addition pathway. Mechanistically, LRP4 functions via the conserved kinase SRPK79D to ensure normal synapse number and behavior. This highlights a presynaptic function for LRP4, enabling deeper understanding of how synapse organization is coordinated.

Wednesday, August 16th

Moose, D. L., Haase, S. J., Aldrich, B. T. and Lear, B. C. (2017). The narrow abdomen ion channel complex is highly stable and persists from development into adult stages to promote behavioral rhythmicity. Front Cell Neurosci 11: 159. PubMed ID: 28634443
The sodium leak channel Narrow abdomen (NA)/ NALCN is an important component of circadian pacemaker neuronal output. In Drosophila, rhythmic expression of the NA channel regulator Nlf-1 in a subset of adult pacemaker neurons has been proposed to contribute to circadian regulation of channel localization or activity. This study restricted expression of Drosophila NA channel subunits or the Nlf-1 regulator to either development or adulthood using the temperature-inducible tubulin-GAL80ts system. Surprisingly, it was found that developmental expression of endogenous channel subunits and Nlf-1 is sufficient to promote robust rhythmic behavior in adults. Moreover, channel complex proteins produced during development persist in the Drosophila head with little decay for at least 5-7 days in adults. In contrast, restricting either endogenous or transgenic gene expression to adult stages produces only limited amounts of the functional channel complex. These data indicate that much of the NA channel complex that functions in adult circadian neurons is normally produced during development, and that the channel complex is very stable in most neurons in the Drosophila brain. Based on these findings, it is proposed that circadian regulation of NA channel function in adult pacemaker neurons is mediated primarily by post-translational mechanisms that are independent of Nlf-1.
Seki, Y., Dweck, H. K. M., Rybak, J., Wicher, D., Sachse, S. and Hansson, B. S. (2017). Olfactory coding from the periphery to higher brain centers in the Drosophila brain. BMC Biol 15(1): 56. PubMed ID: 28666437
Odor information is processed through multiple receptor-glomerular channels in the first order olfactory center, the antennal lobe (AL), then reformatted into higher brain centers and eventually perceived by the fly. To reveal the logic of olfaction, it is fundamental to map odor representations from the glomerular channels into higher brain centers. This study characterized odor response profiles of AL projection neurons (PNs) originating from 31 glomeruli using whole cell patch-clamp recordings in Drosophila melanogaster. Odor representation from olfactory sensory neurons to PNs is generally conserved, while transformation of odor tuning curves is glomerulus-dependent. Reconstructions of PNs reveal that attractive and aversive odors are represented in different clusters of glomeruli in the AL. These separate representations are preserved into higher brain centers, where attractive and aversive odors are segregated into two regions in the lateral horn and partly separated in the mushroom body calyx. This study reveals spatial representation of odor valence coding from the AL to higher brain centers. These results provide a global picture of the olfactory circuit design underlying innate odor-guided behavior.
Placais, P. Y., de Tredern, E., Scheunemann, L., Trannoy, S., Goguel, V., Han, K. A., Isabel, G. and Preat, T. (2017). Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory. Nat Commun 8: 15510. PubMed ID: 28580949
Efficient energy use has constrained the evolution of nervous systems. However, it is unresolved whether energy metabolism may resultantly regulate major brain functions. The observation that Drosophila flies double their sucrose intake at an early stage of long-term memory formation initiated the investigation of how energy metabolism intervenes in this process. Cellular-resolution imaging of energy metabolism reveals a concurrent elevation of energy consumption in neurons of the mushroom body, the fly's major memory centre. Strikingly, upregulation of mushroom body energy flux is both necessary and sufficient to drive long-term memory formation. This effect is triggered by a specific pair of dopaminergic neurons afferent to the mushroom bodies, via the D5-like DAMB dopamine receptor. Hence, dopamine signalling mediates an energy switch in the mushroom body that controls long-term memory encoding. These data thus point to an instructional role for energy flux in the execution of demanding higher brain functions.
Selcho, M., Millan, C., Palacios-Munoz, A., Ruf, F., Ubillo, L., Chen, J., Bergmann, G., Ito, C., Silva, V., Wegener, C. and Ewer, J. (2017). Central and peripheral clocks are coupled by a neuropeptide pathway in Drosophila. Nat Commun 8: 15563. PubMed ID: 28555616
circadian clocks consist of central and peripheral pacemakers, which are coordinated to produce daily rhythms in physiology and behaviour. Despite its importance for optimal performance and health, the mechanism of clock coordination is poorly understood. This study dissected the pathway through which the circadian clock of Drosophila imposes daily rhythmicity to the pattern of adult emergence. Rhythmicity depends on the coupling between the brain clock and a peripheral clock in the prothoracic gland (PG), which produces the steroid hormone, ecdysone. Time information from the central clock is transmitted via the neuropeptide, sNPF, to non-clock neurons that produce the neuropeptide, PTTH. These secretory neurons then forward time information to the PG clock. The central clock exerts a dominant role on the peripheral clock. This use of two coupled clocks could serve as a paradigm to understand how daily steroid hormone rhythms are generated in animals.

Tuesday, August 15th

Parsons, L. M., Grzeschik, N. A., Amaratunga, K., Burke, P., Quinn, L. M. and Richardson, H. E. (2017). A kinome RNAi screen in Drosophila identifies novel genes interacting with Lgl, aPKC and Crb cell polarity genes in epithelial tissues. G3 (Bethesda) 7(8):2497-2509. PubMed ID: 28611255
In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, a boutique collection of RNAi stocks targeting the kinome was screened for their capacity to modify Drosophila 'cell polarity' eye and wing phenotypes. Initially kinase or phosphatase genes were identified whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). Next a secondary screen was conducted to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, JNK, and Notch signalling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways, not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras and lipid/Phospho-inositol-3-kinase (PI3K) signalling pathways. Additionally, it was demonstrated that the 'nutrient sensing' kinases, Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, this screen has revealed novel cell-polarity interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development.
Sap, K. A., Bezstarosti, K., Dekkers, D. H., Voets, O. and Demmers, J. A. (2017). Quantitative proteomics reveals extensive changes in the ubiquitinome after perturbation of the proteasome by targeted dsRNA mediated subunit knockdown in Drosophila. J Proteome Res. PubMed ID: 28665616
The ubiquitin-proteasome system (UPS), a highly regulated mechanism including the active marking of proteins by ubiquitin in order to be degraded, is critical in regulating proteostasis. Dysfunctioning of the UPS has been implicated in diseases such as cancer and neurodegenerative disorders. This study investigated the effects of proteasome malfunctioning on global proteome and ubiquitinome dynamics using SILAC proteomics in Drosophila S2 cells. dsRNA mediated knockdown of specific proteasome target subunits is used to inactivate the proteasome. Upon this perturbation, both the global proteome and the ubiquitinome become modified to a great extent, the overall impact on the ubiquitinome being most dramatic. The abundances of approx. 10% of all proteins are increased, while the abundances of the far majority of over 14 thousand detected diGly peptides are increased, suggesting that the pool of ubiquitinated proteins is highly dynamic. Remarkably, several proteins show heterogeneous ubiquitination dynamics, with different lysine residues on the same protein showing either increased or decreased ubiquitination. This suggests the occurrence of simultaneous and functionally different ubiquitination events. This strategy offers a powerful tool to study the response of the ubiquitinome upon interruption of normal UPS activity by targeted interference and opens up new avenues for the dissection of the mode of action of individual components of the proteasome. Since this is the first comprehensive ubiquitinome screen upon proteasome malfunctioning in a fruit fly cell system, this data set will serve as a valuable repository for the Drosophila community.
Ma, X., Lu, J. Y., Dong, Y., Li, D., Malagon, J. N. and Xu, T. (2017). PP6 disruption synergizes with oncogenic Ras to promote JNK-dependent tumor growth and invasion. Cell Rep 19(13): 2657-2664. PubMed ID: 28658615
RAS genes are frequently mutated in cancers, yet an effective treatment has not been developed, partly because of an incomplete understanding of signaling within Ras-related tumors. To address this, a genetic screen was performed in Drosophila, aiming to find mutations that cooperate with oncogenic Ras (RasV12) to induce tumor overgrowth and invasion. fiery mountain (fmt; CG10289), a regulatory subunit of the protein phosphatase 6 (PP6) complex, was identified as a tumor suppressor that synergizes with RasV12 to drive c-Jun N-terminal kinase (JNK)-dependent tumor growth and invasiveness. Fmt was shown to negatively regulate JNK upstream of dTAK1. It was further demonstrated that disruption of PpV, the catalytic subunit of PP6, mimics fmt loss-of-function-induced tumorigenesis. Finally, Fmt synergizes with PpV to inhibit JNK-dependent tumor progression. These data here further highlight the power of Drosophila as a model system to unravel molecular mechanisms that may be relevant to human cancer biology.
Manavalan, M. A., Jayasinghe, V. R., Grewal, R. and Bhat, K. M. (2017). The glycosylation pathway is required for the secretion of Slit and for the maintenance of the Slit receptor Robo on axons. Sci Signal 10(484). PubMed ID: 28634210
Slit proteins act as repulsive axon guidance cues by activating receptors of the Roundabout (Robo) family. During early neurogenesis in Drosophila melanogaster, Slit prevents the growth cones of longitudinal tract neurons from inappropriately crossing the midline, thus restricting these cells to trajectories parallel to the midline. Slit is expressed in midline glial cells, and Robo is present in longitudinal axon tracts and growth cones. This study shows that the enzyme Mummy (Mmy) controls Slit-Robo signaling through mechanisms that affected both the ligand and the receptor. Mmy was required for the glycosylation of Slit, which was essential for Slit secretion. Mmy was also required for maintaining the abundance and spatial distribution of Robo through an indirect mechanism that was independent of Slit secretion. Moreover, secretion of Slit was required to maintain the fasciculation and position of longitudinal axon tracts, thus maintaining the hardwiring of the nervous system. Thus, Mmy is required for Slit secretion and for maintaining Robo abundance and distribution in the developing nervous system in Drosophila.
Li, C., Li, B., Ma, S., Lu, P. and Chen, K. (2017). Dusky works upstream of Four-jointed and Forked in wing morphogenesis in Tribolium castaneum. Insect Mol Biol. PubMed ID: 28677915
Dusky (dy) is required for cytoskeletal reorganization during wing morphogenesis in Drosophila melanogaster, but which genes participate together with dy for wing morphogenesis has remained unclear. In Tribolium castaneum, dy is highly expressed at the late embryonic stage. Tissue-specific expression analysis indicated high expression levels of dy in the epidermis, head and fat body of late-stage larvae. RNA interference (RNAi) targeting dy significantly decreased adult wing size and caused improper folding of the elytra. Meanwhile, dy knockdown reduced the transcription of four-jointed (fj) and forked (f). These results show that fj RNAi reduces adult wing size and that silencing f results in abnormal wing folding in T. castaneum. Interestingly, knocking down fj and f simultaneously phenocopies dy RNAi, suggesting that dy probably acts upstream of fj and f to regulate wing morphogenesis in T. castaneum.
Rives-Quinto, N., Franco, M., de Torres-Jurado, A. and Carmena, A. (2017). canoe and scribble loss synergizes causing tumor-like overgrowth via Ras activation in neural stem cells and epithelia. Development [Epub ahead of print]. PubMed ID: 28619817
Over the past decade an intriguing connection between asymmetric cell division, stem cells and tumorigenesis has emerged. Neuroblasts, the neural stem cells of the Drosophila central nervous system, divide asymmetrically and constitute an excellent paradigm for further investigating that connection. This study shows that the simultaneous loss of the asymmetric cell division regulators Canoe (Afadin in mammals) and Scribble in neuroblast clones leads to tumor-like overgrowth through both a severe disruption of the asymmetric cell division process and a canoe loss-mediated Ras-PI3K-Akt activation. Moreover, canoe loss also interacts synergistically with scribble to promote overgrowth in epithelial tissues, here just by activating the Ras-Raf-MAPK pathway. Finally scribble functionally related genes discs large and lethal (2) giant larvae were shown to contribute to repress the Ras-MAPK signaling cascade in epithelia. Hence, this work uncovers novel cooperative interactions between all these well-conserved tumor suppressors to ensure a tight regulation of the Ras signaling pathway.

Monday, August 14th

Murata, S., Brockmann, A. and Tanimura, T. (2017). Pharyngeal stimulation with sugar triggers local searching behavior in Drosophila. J Exp Biol [Epub ahead of print]. PubMed ID: 28684466
Foraging behavior is essential for all organisms to find food containing nutritional chemicals. A hungry fly of Drosophila melanogaster performs local searching behavior after drinking a small amount of sugar solution. Using video tracking this study examined how the searching behavior is regulated in D. melanogaster. A small amount of highly concentrated sugar solution was found to induce a long-lasting searching behavior. After the intake of sugar solution, a fly moved around in circles and repeatedly returned to the position where the sugar droplet had been placed. The non-nutritious sugar, D-arabinose, but not the non-sweet nutritious sugar, D-sorbitol, was effective in inducing the behavior, indicating that sweet sensation is essential. Furthermore, pox-neuro mutant flies with no external taste bristles showed local searching behavior, suggesting the involvement of the pharyngeal taste organ. Experimental activation of pharyngeal sugar-sensitive gustatory receptor neurons by capsaicin using the Gal4/UAS system induced local searching behavior. In contrast, inhibition of pharyngeal sugar-responsive gustatory receptor neurons abolished the searching behavior. Together these results indicate that in Drosophila the pharyngeal taste-receptor neurons trigger searching behavior immediately after ingestion.
McConnell, M. W. and Fitzpatrick, M. J. (2017). 'Foraging' for a place to lay eggs: A genetic link between foraging behaviour and oviposition preferences. PLoS One 12(6): e0179362. PubMed ID: 28622389
Gravid female arthropods in search of egg-laying substrates embark on foraging-like forays: they survey the environment assessing multiple patches, tasting each with their tarsi and proboscis, and then, if interested, they deposit an egg (or eggs). In fruit flies, Drosophila melanogaster, allelic variation in the foraging gene (for) underlies the rover/sitter foraging behaviour polymorphism. Rover flies (forR) are more active foragers (both within and between food patches) compared to sitters (fors). In nematodes, Caenorhabditis elegans, a mutation in egl-4, the ortholog of for, leads to aberrations in egg laying. Given this and the notion that females may 'forage' for a place to oviposit, it was hypothesized that for may underlie egg-laying decisions in the fruit fly. Indeed, when given a choice between patches of low- and high-nutrient availability, rovers lay significantly more eggs on the low-nutrient patches than sitters and also a sitter mutant (fors2). This study confirmed the role of for by inducing rover-like oviposition preferences in a sitter fly using the transgenic overexpression of for-mRNA in the nervous system.
Liang, X., Holy, T. E. and Taghert, P. H. (2017). A series of suppressive signals within the Drosophila circadian neural circuit generates sequential daily outputs. Neuron 94(6): 1173-1189.e1174. PubMed ID: 28552314
The Drosophila circadian neural circuit was studied using whole-brain imaging in vivo. Five major groups of pacemaker neurons display synchronized molecular clocks, yet each exhibits a distinct phase of daily Ca2+ activation. Light and neuropeptide pigment dispersing factor (PDF) from morning cells (s-LNv) together delay the phase of the evening (LNd) group by approximately 12 hr; PDF alone delays the phase of the DN3 group by approximately 17 hr. Neuropeptide sNPF, released from s-LNv and LNd pacemakers, produces Ca2+ activation in the DN1 group late in the night. The circuit also features negative feedback by PDF to truncate the s-LNv Ca2+ wave and terminate PDF release. Both PDF and sNPF suppress basal Ca2+ levels in target pacemakers with long durations by cell-autonomous actions. Thus, light and neuropeptides act dynamically at distinct hubs of the circuit to produce multiple suppressive events that create the proper tempo and sequence of circadian pacemaker neuronal activities.
Chouhan, N. S., Wolf, R. and Heisenberg, M. (2017). Starvation promotes odor/feeding-time associations in flies. Learn Mem 24(7): 318-321. PubMed ID: 28620079
Starvation causes a motivational state that facilitates diverse behaviors such as feeding, walking, and search. Starved Drosophila can form odor/feeding-time associations but the role of starvation in encoding of "time" is poorly understood. This study shows that the extent of starvation is correlated with the fly's ability to establish odor/feeding-time memories. Prolonged starvation promotes odor/feeding-time associations after just a single cycle of reciprocal training. Starvation is also show to be required for acquisition but is dispensable for retrieval of odor/feeding-time memory. Finally, even with extended starvation, a functional circadian oscillator is indispensable for establishing odor/feeding-time memories.

Sunday, August 13th

Yenigun, V. B., Sirito, M., Amcheslavky, A., Czernuszewicz, T., Colonques-Bellmunt, J., Garcia-Alcover, I., Wojciechowska, M., Bolduc, C., Chen, Z., Lopez Castel, A., Krahe, R. and Bergmann, A. (2017). (CCUG)n RNA toxicity in a Drosophila model for myotonic dystrophy type 2 (DM2) activates apoptosis. Dis Model Mech. PubMed ID: 28623239
The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions -- (CTG)DM1 in DMPK and (CCTG)DM2 in CNBP. Although transcription of mutant repeats into (CUG)DM1 or (CCUG)DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG)DM2 toxicity and develop a convenient model for drug screening, a transgenic DM2 model was developed in the fruit fly Drosophila melanogaster with (CCUG)n repeats of variable length (n=16 and 106). Expression of noncoding (CCUG)106, but not (CCTG)16, in muscle and retinal cells led to formation of (CCUG) ribonuclear inclusions and mis-splicing of genes implicated in the DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, while CUGBP1/CELF1 complementation did not. Flies with (CCUG)106 displayed strong disruption of the external eye morphology and the underlying retina. Furthermore, expression of (CCUG)106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG)106 flies. Finally, two chemical compounds were tested that have shown therapeutic potential in DM1 models. While treatment of (CCUG)106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both formation of RNA foci and apoptosis in retinae of (CCUG)106 flies. These data indicate that expression of expanded (CCUG)DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. The Drosophila DM2 model may provide a convenient tool for in vivo drug screening.
M'Angale, P. G. and Staveley, B. E. (2017). A loss of Pdxk model of Parkinson disease in Drosophila can be suppressed by Buffy. BMC Res Notes 10(1): 205. PubMed ID: 28606139
The identification of a DNA variant in pyridoxal kinase (Pdxk) associated with increased risk to Parkinson disease (PD) gene has led to a study the inhibition of this gene in the Dopa decarboxylase (Ddc)-expressing neurons of Drosophila. The multitude of biological functions attributable to the vitamers of vitamin B6 catalysed by this kinase reveal an overabundance of possible links to PD, that include dopamine synthesis, antioxidant activity and mitochondrial function. Drosophila possesses a single homologue of Pdxk, and this study used RNAi to inhibit the activity of this kinase in the Ddc-Gal4-expressing neurons. Any association was further investigated between this enhanced disease risk gene with the established PD model induced by expression of alpha-synuclein in the same neurons. The pro-survival functions of Buffy, an anti-apoptotic Bcl-2 homologue, were relied on to rescue the Pdxk-induced phenotypes. Ddc-Gal4, which drives expression in both dopaminergic and serotonergic neurons, was used to drive the expression of Pdxk RNA interference in DA neurons of Drosophila. The inhibition of Pdxk in the alpha-synuclein-induced Drosophila model of PD did not alter longevity and climbing ability of these flies. It has been previously shown that deficiency in vitamers lead to mitochondrial dysfunction and neuronal decay, therefore, co-expression of Pdxk-RNAi with the sole pro-survival Bcl-2 homologue Buffy in the Ddc-Gal4-expressing neurons, resulted in increased survival and a restored climbing ability. In a similar manner, when Pdxk was inhibited in the developing eye using GMR-Gal4, it was found that there was a decrease in the number of ommatidia and the disruption of the ommatidial array was more pronounced. When Pdxk was inhibited with the alpha-synuclein-induced developmental eye defects, the eye phenotypes were unaltered. Interestingly co-expression with Buffy restored ommatidia number and decreased the severity of disruption of the ommatidial array. It is concluded that though Pdxk is not a confirmed Parkinson disease gene, the inhibition of this kinase recapitulated the PD-like symptoms of decreased lifespan and loss of locomotor function, possibly producing a new model of PD.
Meng, H., Yamashita, C., Shiba-Fukushima, K., Inoshita, T., Funayama, M., Sato, S., Hatta, T., Natsume, T., Umitsu, M., Takagi, J., Imai, Y. and Hattori, N. (2017). Loss of Parkinson's disease-associated protein CHCHD2 affects mitochondrial crista structure and destabilizes Cytochrome c. Nat Commun 8: 15500. PubMed ID: 28589937
Mutations in CHCHD2 have been identified in some Parkinson's disease (PD) cases. To understand the physiological and pathological roles of CHCHD2, this study manipulated the expression of CHCHD2 in Drosophila and mammalian cells. The loss of CHCHD2 in Drosophila causes abnormal matrix structures and impaired oxygen respiration in mitochondria, leading to oxidative stress, dopaminergic neuron loss and motor dysfunction with age. These PD-associated phenotypes are rescued by the overexpression of the translation inhibitor 4E-BP and by the introduction of human CHCHD2 but not its PD-associated mutants. CHCHD2 is upregulated by various mitochondrial stresses, including the destabilization of mitochondrial genomes and unfolded protein stress, in Drosophila. CHCHD2 binds to cytochrome c along with a member of the Bax inhibitor-1 superfamily, MICS1, and modulated cell death signalling, suggesting that CHCHD2 dynamically regulates the functions of cytochrome c in both oxidative phosphorylation and cell death in response to mitochondrial stress.
Hosaka, Y., Inoshita, T., Shiba-Fukushima, K., Cui, C., Arano, T., Imai, Y. and Hattori, N. (2017). Reduced TDP-43 expression improves neuronal activities in a Drosophila model of Perry syndrome. EBioMedicine [Epub ahead of print]. PubMed ID: 28625517
Parkinsonian Perry syndrome, involving mutations in the dynein motor component dynactin or p150Glued, is characterized by TDP-43 pathology in affected brain regions, including the substantia nigra. However, the molecular relationship between p150Glued and TDP-43 is largely unknown. This study reports that a reduction in TDP-43 protein levels alleviates the synaptic defects of neurons expressing the Perry mutant p150G50R in Drosophila. Dopaminergic expression of p150G50R, which decreases dopamine release, disrupts motor ability and reduces the lifespan of Drosophila. p150G50R expression also causes aggregation of dense core vesicles (DCVs), which contain monoamines and neuropeptides, and disrupts the axonal flow of DCVs, thus decreasing synaptic strength. The above phenotypes associated with Perry syndrome are improved by the removal of a copy of Drosophila TDP-43 TBPH, thus suggesting that the stagnation of axonal transport by dynactin mutations promotes TDP-43 aggregation and interferes with the dynamics of DCVs and synaptic activities.

Saturday, August 12th

Bataille, L., Boukhatmi, H., Frendo, J. L. and Vincent, A. (2017). Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles. BMC Biol 15(1): 48. PubMed ID: 28599653
A stereotyped array of body wall muscles enables precision and stereotypy of animal movements. In Drosophila, each syncytial muscle forms via fusion of one founder cell (FC) with multiple fusion competent myoblasts (FCMs). The specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors (iTFs), such as Apterous, Even-Skipped and Slouch/S59, expressed by its FC. This study addressed three questions: Are FCM nuclei naive? What is the selectivity and temporal sequence of transcriptional reprogramming of FCMs recruited into growing syncytium? Is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation? The tracking of nuclei in developing muscles shows that FCM nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. In situ hybridisation to nascent transcripts for FCM, FC-generic and iTF genes shows that this reprogramming is progressive, beginning by repression of FCM-specific genes in fused nuclei, with some evidence that FC nuclei retain specific characteristics. Transcription of identity realisation genes is linked to iTF activation and regulated at levels of both transcription initiation rate and period of transcription. The generic muscle differentiation programme is activated independently. It is concluded that transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. This comprehensive view of the dynamics of transcriptional (re)programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells.
Sallee, M. D., Littleford, H. E. and Greenwald, I. (2017). A bHLH code for sexually dimorphic form and function of the C. elegans somatic gonad. Curr Biol 27(12): 1853-1860 e1855. PubMed ID: 28602651
Evolutionary Homolog Study
How sexually dimorphic gonads are generated is a fundamental question at the interface of developmental and evolutionary biology. In C. elegans, sexual dimorphism in gonad form and function largely originates in different apportionment of roles to three regulatory cells of the somatic gonad primordium in young larvae. Their essential roles include leading gonad arm outgrowth, serving as the germline niche, connecting to epithelial openings, and organizing reproductive organ development. The development and function of the regulatory cells in both sexes requires the basic-helix-loop-helix (bHLH) transcription factor HLH-2, the sole ortholog of the E proteins mammalian E2A and Drosophila Daughterless, yet how they adopt different fates to execute their different roles has been unknown. This study shows that each regulatory cell expresses a distinct complement of bHLH-encoding genes-and therefore distinct HLH-2:bHLH dimers-and formulate a "bHLH code" hypothesis for regulatory cell identity. This hypothesis is supported by showing that the bHLH gene complement is both necessary and sufficient to confer particular regulatory cell fates. Strikingly, prospective regulatory cells can be directly reprogrammed into other regulatory cell types simply by loss or ectopic expression of bHLH genes, and male-to-female and female-to-male transformations indicate that the code is instructive for sexual dimorphism. The bHLH code appears to be embedded in a bow-tie regulatory architecture, wherein sexual, positional, temporal, and lineage inputs connect through bHLH genes to diverse outputs for terminal features and provides a plausible mechanism for the evolutionary plasticity of gonad form seen in nematodes.
Kushnir, T., Mezuman, S., Bar-Cohen, S., Lange, R., Paroush, Z. and Helman, A. (2017). Novel interplay between JNK and Egfr signaling in Drosophila dorsal closure. PLoS Genet 13(6): e1006860. PubMed ID: 28628612
Dorsal closure (DC) is a developmental process in which two contralateral epithelial sheets migrate to seal a large hole in the dorsal ectoderm of the Drosophila embryo. Two signaling pathways act sequentially to orchestrate this dynamic morphogenetic process. First, c-Jun N-terminal kinase (JNK) signaling activity in the dorsal-most leading edge (LE) cells of the epidermis induces expression of decapentaplegic (dpp). Second, Dpp, a secreted TGF-beta homolog, triggers cell shape changes in the adjacent, ventrally located lateral epidermis, that guide the morphogenetic movements and cell migration mandatory for DC. This study uncovered a cell non-autonomous requirement for the Epidermal growth factor receptor (Egfr) pathway in the lateral epidermis for sustained dpp expression in the LE. Specifically, it was demonstrated that Egfr pathway activity in the lateral epidermis prevents expression of the gene scarface (scaf), encoding a secreted antagonist of JNK signaling. In embryos with compromised Egfr signaling, upregulated Scaf causes reduction of JNK activity in LE cells, thereby impeding completion of DC. These results identify a new developmental role for Egfr signaling in regulating epithelial plasticity via crosstalk with the JNK pathway.
Olivares-Castineira, I. and Llimargas, M. (2017). EGFR controls Drosophila tracheal tube elongation by intracellular trafficking regulation. PLoS Genet 13(7): e1006882. PubMed ID: 28678789
Development is governed by a few conserved signalling pathways. Amongst them, the EGFR pathway is used reiteratively for organ and tissue formation, and when dysregulated can lead to cancer and metastasis. Given its relevance, identifying its downstream molecular machinery and understanding how it instructs cellular changes is crucial. This study approached this issue in the respiratory system of Drosophila. A new role was identified for EGFR restricting the elongation of the tracheal Dorsal Trunk. EGFR was found to regulate the apical determinant Crumbs and the extracellular matrix regulator Serpentine, two factors previously known to control tube length. EGFR regulates the organisation of endosomes in which Crb and Serp proteins are loaded. These results are consistent with a role of EGFR in regulating Retromer/WASH recycling routes. Furthermore, this study provides new insights into Crb trafficking and recycling during organ formation. This work connects cell signalling, trafficking mechanisms and morphogenesis and suggests that the regulation of cargo trafficking can be a general outcome of EGFR activation.

Friday, August 11th

Obadia, B., Guvener, Z. T., Zhang, V., Ceja-Navarro, J. A., Brodie, E. L., Ja, W. W. and Ludington, W. B. (2017). Probabilistic invasion underlies natural gut microbiome stability. Curr Biol [Epub ahead of print]. PubMed ID: 28625783
Species compositions of gut microbiomes impact host health, but the processes determining these compositions are largely unknown. An unexplained observation is that gut species composition varies widely between individuals but is largely stable over time within individuals. Stochastic factors during establishment may drive these alternative stable states (colonized versus non-colonized), which can influence susceptibility to pathogens, such as Clostridium difficile. A precise, high-throughput technique revealed stable between-host variation in colonization when individual germ-free flies were fed their own natural commensals (including the probiotic Lactobacillus plantarum). Some flies were colonized while others remained germ-free even at extremely high bacterial doses. Thus, alternative stable states of colonization exist even in this low-complexity model of host-microbe interactions. These alternative states are driven by a fundamental asymmetry between the inoculum population and the stably colonized population that is mediated by spatial localization and a population bottleneck, which makes stochastic effects important by lowering the effective population size. Prior colonization with other bacteria reduced the chances of subsequent colonization, thus increasing the stability of higher-diversity guts. Therefore, stable gut diversity may be driven by inherently stochastic processes, which has important implications for combatting infectious diseases and for stably establishing probiotics in the gut.
Garcia, J. F., Carbone, M. A., Mackay, T. F. C. and Anholt, R. R. H. (2017). Regulation of Drosophila lifespan by bellwether promoter alleles. Sci Rep 7(1): 4109. PubMed ID: 28646164
Longevity varies among individuals, but how natural genetic variation contributes to variation in lifespan is poorly understood. Drosophila melanogaster presents an advantageous model system to explore the genetic underpinnings of longevity, since its generation time is brief and both the genetic background and rearing environment can be precisely controlled. The bellwether (blw) gene encodes the alpha subunit of mitochondrial ATP synthase. Since metabolic rate may influence lifespan, this study investigated whether alternative haplotypes in the blw promoter affect lifespan when expressed in a co-isogenic background. 521 bp upstream promoter sequences containing the alternative SNP haplotypes (G/T and A/G) were amplified, and promoter activity was assessed both in vitro and in vivo using a luciferase reporter system. The AG haplotype showed significantly greater expression of luciferase than the GT haplotype. A blw cDNA construct driven by either the AG or GT haplotype promoter was driven in transgenic flies, and the AG haplotype was shown to results in greater blw cDNA expression and a significant decrease in lifespan relative to the GT promoter haplotype, in male flies only. Thus, the results show that naturally occurring regulatory variants of blw affect lifespan in a sex-specific manner.
Hazegh, K. E., Nemkov, T., D'Alessandro, A., Diller, J. D., Monks, J., McManaman, J. L., Jones, K. L., Hansen, K. C. and Reis, T. (2017). An autonomous metabolic role for Spen. PLoS Genet 13(6): e1006859. PubMed ID: 28640815
Preventing obesity requires a precise balance between deposition into and mobilization from fat stores, but regulatory mechanisms are incompletely understood. Drosophila Split ends (Spen) is the founding member of a conserved family of RNA-binding proteins involved in transcriptional regulation and frequently mutated in human cancers. This study found that manipulating Spen expression alters larval fat levels in a cell-autonomous manner. Spen-depleted larvae had defects in energy liberation from stores, including starvation sensitivity and major changes in the levels of metabolic enzymes and metabolites, particularly those involved in beta-oxidation. Spenito, a small Spen family member, counteracted Spen function in fat regulation. Finally, mouse Spen and Spenito transcript levels scaled directly with body fat in vivo, suggesting a conserved role in fat liberation and catabolism. This study demonstrates that Spen is a key regulator of energy balance and provides a molecular context to understand the metabolic defects that arise from Spen dysfunction.
Garcia, C. J., Khajeh, J., Coulanges, E., Chen, E. I. and Owusu-Ansah, E. (2017). Regulation of mitochondrial complex I biogenesis in Drosophila flight muscles. Cell Rep 20(1): 264-278. PubMed ID: 28683319
The flight muscles of Drosophila are highly enriched with mitochondria, but the mechanism by which mitochondrial complex I (CI) is assembled in this tissue has not been described. This study reports the mechanism of CI biogenesis in Drosophila flight muscles and shows that it proceeds via the formation of approximately 315, approximately 550, and approximately 815 kDa CI assembly intermediates. Additionally, specific roles were defined for several CI subunits in the assembly process. In particular, dNDUFS5 was shown to be required for converting an approximately 700 kDa transient CI assembly intermediate into the approximately 815 kDa assembly intermediate. Importantly, incorporation of dNDUFS5 into CI is necessary to stabilize or promote incorporation of dNDUFA10 into the complex. These findings highlight the potential of studies of CI biogenesis in Drosophila to uncover the mechanism of CI assembly in vivo and establish Drosophila as a suitable model organism and resource for addressing questions relevant to CI biogenesis in humans.

Thursday, August 10th

Moshe, A. and Kaplan, T. (2017). Genome-wide search for Zelda-like chromatin signatures identifies GAF as a pioneer factor in early fly development. Epigenetics Chromatin 10(1): 33. PubMed ID: 28676122
The protein Zelda was shown to play a key role in early Drosophila development, binding thousands of promoters and enhancers prior to maternal-to-zygotic transition (MZT), and marking them for transcriptional activation. Zelda has been shown to act through specific chromatin patterns of histone modifications to mark developmental enhancers and active promoters. Intriguingly, some Zelda sites still maintain these chromatin patterns in Drosophila embryos lacking maternal Zelda protein. This suggests that additional Zelda-like pioneer factors may act in early fly embryos. A computational method was developed to analyze and refine the chromatin landscape surrounding early Zelda peaks, using a multichannel spectral clustering. This allowed characterization their chromatin patterns through MZT (mitotic cycles 8-14). Specifically, focus was placed on H3K4me1, H3K4me3, H3K18ac, H3K27ac, and H3K27me3 and three different classes of chromatin signatures were identified, matching "promoters," "enhancers" and "transiently bound" Zelda peaks. Then the genome was further scanned using these chromatin patterns and additional loci - with no Zelda binding- were identified that show similar chromatin patterns, resulting with hundreds of Zelda-independent putative enhancers. These regions were found to be enriched with GAGA factor (GAF, Trl) and are typically located near early developmental zygotic genes. Overall this analysis suggests that GAF, together with Zelda, plays an important role in activating the zygotic genome. The computational approach offers an efficient algorithm for characterizing chromatin signatures around some loci of interest and allows a genome-wide identification of additional loci with similar chromatin patterns.
Martin, R. L., Maiorano, J., Beitel, G. J., Marko, J. F., McVicker, G. and Fondufe-Mittendorf, Y. N. (2017). A comparison of nucleosome organization in Drosophila cell lines. PLoS One 12(6): e0178590. PubMed ID: 28570602
Changes in the distribution of nucleosomes along the genome influence chromatin structure and impact gene expression by modulating the accessibility of DNA to transcriptional machinery. This study compared genome-wide nucleosome positioning and occupancy in five different Drosophila tissue-specific cell lines, and in reconstituted chromatin, and then tests were performed for correlations between nucleosome positioning, transcription factor binding motifs, and gene expression. Nucleosomes in all cell lines were positioned in accordance with previously known DNA-nucleosome interactions, with helically repeating A/T di-nucleotide pairs arranged within nucleosomal DNAs and AT-rich pentamers generally excluded from nucleosomal DNA. Nucleosome organization in all cell lines differed markedly from in vitro reconstituted chromatin, with highly expressed genes showing strong nucleosome organization around transcriptional start sites. Importantly, comparative analysis identified genomic regions that exhibited cell line-specific nucleosome enrichment or depletion. Further analysis of these regions identified 91 out of 16,384 possible heptamer sequences that showed differential nucleosomal occupation between cell lines, and 49 of the heptamers matched one or more known transcription factor binding sites. These results demonstrate that there is differential nucleosome positioning between these Drosophila cell lines and therefore identify a system that could be used to investigate the functional significance of differential nucleosomal positioning in cell type specification.
Jox, T., Buxa, M. K., Bohla, D., Ullah, I., Macinkovic, I., Brehm, A., Bartkuhn, M. and Renkawitz, R. (2017). Drosophila CP190- and dCTCF-mediated enhancer blocking is augmented by SUMOylation. Epigenetics Chromatin. 10: 32. PubMed ID: 28680483d

Chromatin insulators shield promoters and chromatin domains from neighboring enhancers or chromatin regions with opposing activities. Insulator-binding proteins and their cofactors mediate the boundary function. In general, covalent modification of proteins by the small ubiquitin-like modifier (SUMO) is an important mechanism to control the interaction of proteins within complexes. This study addressed the impact of dSUMO in respect of insulator function, chromatin binding of insulator factors and formation of insulator speckles in Drosophila. SUMOylation augments the enhancer blocking function of four different insulator sequences and increases the genome-wide binding of the insulator cofactor CP190. These results indicate that enhanced chromatin binding of SUMOylated CP190 causes fusion of insulator speckles, which may allow for more efficient insulation.

Dutta, P. and Li, W. X. (2017). The SERTAD protein Taranis plays a role in Polycomb-mediated gene repression. PLoS One 12(6): e0180026. PubMed ID: 28665982

The Polycomb group (PcG) proteins have been implicated in epigenetic transcriptional repression in development, stem cell maintenance and in cancer. The chromodomain protein Polycomb (Pc) is a key member of the PcG. Pc binds to the histone mark, trimethylated histone 3 lysine 27 (H3K27me3), to initiate transcriptional repression. How PcG proteins are recruited to target loci is not fully understood. This study shows that the Drosophila SERTA domain protein Taranis (Tara) is involved in transcriptional regulation of Pc target genes. Embryos lacking Tara exhibit a partial homeotic transformation of cuticular the segments, a phenotype associated with the loss of Pc function. Moreover, Drosophila embryos homozygous for a tara hypomorphic allele also misexpress engrailed, a Pc-regulated gene, and this phenotype is associated with the loss of Pc binding to the cis response element in the engrailed enhancer. In relation to that, Pc recruitment is reduced on the salivary gland polytene chromosomes and specifically at the engrailed locus. These results suggest that Tara might be required for positioning Pc to a subset of its target genes.

Wednesday, August 9th

LaFerriere, H. and Zars, T. (2017). The Drosophila melanogaster tribbles pseudokinase is necessary for proper memory formation. Neurobiol Learn Mem 144: 68-76. PubMed ID: 28669782
The tribbles (trbl) pseudokinases play important roles in signaling and physiology in multiple contexts, ranging from innate immunity to cancer, suggesting fundamental cellular functions for the trbl gene products. Despite expression of the trbl pseudokinases in the nervous systems of invertebrate and vertebrate animals, and evidence that they have a function within mouse and human dopamine neurons, there is no clear case for a function of a Trbl protein that influences behavior. Indeed, the first and only evidence for this type of function comes from Drosophila melanogaster, where a mutation of the single trbl gene was identified in a genetic screen for short-term memory mutant flies. The current study tested flies containing multiple trbl mutant alleles and potential transgenic rescue in both operant place memory and classical olfactory memory paradigms. Genetic complementation tests and transgenic rescue of memory phenotypes in both paradigms show that the D. melanogaster trbl pseudokinase is essential for proper memory formation. Expression analysis with a polyclonal antiserum against Trbl shows that the protein is expressed widely in the fly brain, with higher expression in the cellular rind than the neuropil. Rescue of the behavioral phenotype with transgenic expression indicates the trbl function can be localized to a subset of the nervous system. Thus, this study provides the first compelling case for the function of a trbl pseudokinase in the regulation of behavior.
Lark, A., Kitamoto, T. and Martin, J. R. (2017). Modulation of neuronal activity in the Drosophila mushroom body by DopEcR, a unique dual receptor for ecdysone and dopamine. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 28554773
G-protein-coupled receptors (GPCRs) for steroid hormones mediate unconventional steroid signaling. Drosophila DopEcR is a GPCR that responds to both ecdysone (the major steroid hormone in insects) and dopamine, regulating multiple second messenger systems. Recent studies have revealed that DopEcR is preferentially expressed in the nervous system and involved in behavioral regulation. This study utilized the bioluminescent Ca2+-indicator GFP-aequorin to monitor the nicotine-induced Ca2+-response within the mushroom bodies (MB), a higher-order brain center in flies, and examined how DopEcR modulates these Ca2+-dynamics. The results show that in DopEcR knockdown flies, the nicotine-induced Ca2+-response in the MB was significantly enhanced selectively in the medial lobes. Application of DopEcR's ligands, ecdysone and dopamine, had different effects on nicotine-induced Ca2+-responses in the MB: ecdysone enhanced activity in the calyx and cell body region in a DopEcR-dependent manner, whereas dopamine reduced activity in the medial lobes independently of DopEcR. Finally, flies with reduced DopEcR function in the MB were shown to display decreased locomotor activity. This behavioral phenotype of DopEcR-deficient flies may be partly due to their enhanced MB activity, since the MB have been implicated in the suppression of locomotor activity. Overall, these data suggest that DopEcR is involved in region-specific modulation of Ca2+ dynamics within the MB, which may play a role in behavioral modulation.
Lovick, J. K., Omoto, J. J., Ngo, K. T. and Hartenstein, V. (2017). Development of the anterior visual input pathway to the Drosophila central complex. J Comp Neurol. PubMed ID: 28675433
The anterior visual pathway (AVP) conducts visual information from the medulla of the optic lobe via the anterior optic tubercle (AOTU) and bulb (BU) to the ellipsoid body (EB) of the central complex. This paper analyzes the formation of the AVP from early larval to adult stages. The immature fiber tracts of the AVP, formed by secondary neurons of lineages DALcl1/2 and DALv2, assemble into structurally distinct primordia of the AOTU, BU, and EB within the late larval brain. During the early pupal period (P6-P48) these primordia grow in size and differentiate into the definitive subcompartments of the AOTU, BU, and EB. The primordium of the EB has a complex composition. DALv2 neurons form the anterior EB primordium, which starts out as a bilateral structure, then crosses the midline between P6 and P12, and subsequently bends to adopt the ring shape of the mature EB. Columnar neurons of the central complex, generated by the type II lineages DM1-4, form the posterior EB primordium. Starting out as an integral part of the fan-shaped body (FB) primordium, the posterior EB primordium moves forward and merges with the anterior EB primordium. This paper documents the extension of neuropil glia around the nascent EB and BU and analyzes the relationship of primary and secondary neurons of the AVP lineages.
Knecht, Z. A., Silbering, A. F., Cruz, J., Yang, L., Croset, V., Benton, R. and Garrity, P. A. (2017). Ionotropic Receptor-dependent moist and dry cells control hygrosensation in Drosophila. Elife 6. PubMed ID: 28621663
Insects use hygrosensation (humidity sensing) to avoid desiccation and, in vectors such as mosquitoes, to locate vertebrate hosts. Sensory neurons activated by either dry or moist air ('dry cells' and 'moist cells') have been described in many insects, but their behavioral roles and the molecular basis of their hygrosensitivity remain unclear. It has been reported that Drosophila hygrosensation relies on three Ionotropic Receptors (IRs) required for dry cell function: IR25a, IR93a and IR40a. This paper reports the discovery of Drosophila moist cells and shows that they require IR25a and IR93a together with IR68a, a conserved, but orphan IR. Both IR68a- and IR40a-dependent pathways drive hygrosensory behavior: each is important for dry-seeking by hydrated flies and together they underlie moist-seeking by dehydrated flies. These studies reveal that humidity sensing in Drosophila, and likely other insects, involves the combined activity of two molecularly related but neuronally distinct hygrosensing systems.

Tuesday, August 8th

Gonzalez, D. A., et al. (2017). The Arf6 activator Efa6/PSD3 confers regional specificity and modulates ethanol consumption in Drosophila and humans. Mol Psychiatry [Epub ahead of print]. PubMed ID: 28607459
The ubiquitously expressed small GTPase Arf6 is required for normal ethanol-induced sedation in adult Drosophila. This behavioral response also requires Efa6, one of three Drosophila Arf6 guanine exchange factors. Ethanol-naive Arf6 and Efa6 mutants were sensitive to ethanol-induced sedation and lacked rapid tolerance upon re-exposure to ethanol, when compared with wild-type flies. In contrast to wild-type flies, both Arf6 and Efa6 mutants preferred alcohol-containing food without prior ethanol experience. An analysis of the human ortholog of Arf6 and orthologs of Efa6 (PSD1-4) revealed that the minor G allele of single nucleotide polymorphism (SNP) rs13265422 in PSD3, as well as a haplotype containing rs13265422, was associated with an increased frequency of drinking and binge drinking episodes in adolescents. The same haplotype was also associated with increased alcohol dependence in an independent European cohort. Unlike the ubiquitously expressed human Arf6 GTPase, PSD3 localization is restricted to the brain, particularly the prefrontal cortex (PFC). Functional magnetic resonance imaging revealed that the same PSD3 haplotype was also associated with a differential functional magnetic resonance imaging signal in the PFC during a Go/No-Go task, which engages PFC-mediated executive control. This analysis therefore suggests that PSD3 confers regional specificity to ubiquitous Arf6 in the PFC to modulate human alcohol-drinking behaviors.
Hautbergue, G. M., et al. (2017). SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits. Nat Commun 8: 16063. PubMed ID: 28677678
Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. This study shows that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. it was further demonstrated that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, this study shows that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection.
Sekiya, M., Maruko-Otake, A., Hearn, S., Sakakibara, Y., Fujisaki, N., Suzuki, E., Ando, K. and Iijima, K. M. (2017). EDEM function in ERAD protects against chronic ER Proteinopathy and age-related physiological decline in Drosophila. Dev Cell 41(6): 652-664.e655. PubMed ID: 28633019
The unfolded protein response (UPR), which protects cells against accumulation of misfolded proteins in the ER, is induced in several age-associated degenerative diseases. However, sustained UPR activation has negative effects on cellular functions and may worsen disease symptoms. It remains unknown whether and how UPR components can be utilized to counteract chronic ER proteinopathies. This study found that promotion of ER-associated degradation (ERAD) through upregulation of ERAD-enhancing alpha-mannosidase-like proteins [EDEMs; EDEM1 (CG3810) and EDEM2 (CG5682)] protected against chronic ER proteinopathy without inducing toxicity in a Drosophila model. ERAD activity in the brain decreased with aging, and upregulation of EDEMs suppressed age-dependent behavioral decline and extended the lifespan without affecting the UPR gene expression network. Intriguingly, EDEM mannosidase activity was dispensable for these protective effects. Therefore, upregulation of EDEM function in the ERAD protects against ER proteinopathy in vivo and thus represents a potential therapeutic target for chronic diseases.
Lim, N. R., Shohayeb, B., Zaytseva, O., Mitchell, N., Millard, S. S., Ng, D. C. H. and Quinn, L. M. (2017). Glial-specific functions of microcephaly protein WDR62 and interaction with the mitotic kinase AURKA are essential for Drosophila brain growth. Stem Cell Reports [Epub ahead of print]. PubMed ID: 28625535
The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. This study used Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. It was further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA. Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly.

Monday, August 7th

Garcia Garcia, E., Little, J. C. and Kalderon, D. (2017). The exon junction complex and Srp54 contribute to Drosophila Hedgehog signaling via ci RNA splicing. Genetics [Epub ahead of print]. PubMed ID: 28637711
Hedgehog (Hh) regulates the Cubitus interruptus (Ci) transcription factor in Drosophila melanogaster by activating full-length Ci-155 and blocking processing to Ci-75 repressor. However, the interplay between regulation of Ci-155 levels and activity, as well as processing-independent mechanisms that affect Ci-155 levels have not been explored extensively. This study has identified Mago Nashi (Mago) and Y14 core Exon Junction Complex (EJC) proteins, as well as the Srp54 splicing factor as modifiers of Hh pathway activity under sensitized conditions. Mago inhibition reduced Hh pathway activity by altering the splicing pattern of ci to reduce Ci-155 levels. Srp54 inhibition also affected pathway activity by reducing ci RNA levels but additionally altered Ci-155 levels and activity independently of ci splicing. Further tests using ci transgenes and ci mutations confirmed evidence from studying the effects of Mago and Srp54 that relatively small changes in the level of Ci-155 primary translation product alter Hh pathway activity under a variety of sensitized conditions. ci transgenes lacking intron sequences or the presumed translation initiation codon were used for an alternatively spliced ci RNA to provide further evidence that Mago acts principally by modulating the levels of the major ci RNA encoding Ci-155, and to show that ci introns are necessary to support production of sufficient Ci-155 for robust Hh signaling and may also be important mediators of regulatory inputs.
Li, Q., Kellner, D. A., Hatch, H. A. M., Yumita, T., Sanchez, S., Machold, R. P., Frank, C. A. and Stavropoulos, N. (2017). Conserved properties of Drosophila Insomniac link sleep regulation and synaptic function. PLoS Genet 13(5): e1006815. PubMed ID: 28558011
Sleep is an ancient animal behavior that is regulated similarly in species ranging from flies to humans. Various genes that regulate sleep have been identified in invertebrates, but whether the functions of these genes are conserved in mammals remains poorly explored. Drosophila insomniac (inc) mutants exhibit severely shortened and fragmented sleep. Inc protein physically associates with the Cullin-3 (Cul3) ubiquitin ligase, and neuronal depletion of Inc or Cul3 strongly curtails sleep, suggesting that Inc is a Cul3 adaptor that directs the ubiquitination of neuronal substrates that impact sleep. Three proteins similar to Inc exist in vertebrates-KCTD2, KCTD5, and KCTD17-but are uncharacterized within the nervous system and their functional conservation with Inc has not been addressed. This study shows that Inc and its mouse orthologs exhibit striking biochemical and functional interchangeability within Cul3 complexes. Remarkably, KCTD2 and KCTD5 restore sleep to inc mutants, indicating that they can substitute for Inc in vivo and engage its neuronal targets relevant to sleep. Inc and its orthologs localize similarly within fly and mammalian neurons and can traffic to synapses, suggesting that their substrates may include synaptic proteins. Consistent with such a mechanism, inc mutants exhibit defects in synaptic structure and physiology, indicating that Inc is essential for both sleep and synaptic function. These findings reveal that molecular functions of Inc are conserved through ~600 million years of evolution and support the hypothesis that Inc and its orthologs participate in an evolutionarily conserved ubiquitination pathway that links synaptic function and sleep regulation.
Hall, E. T., Pradhan-Sundd, T., Samnani, F. and Verheyen, E. M. (2017). The Protein Phosphatase 4 complex promotes the Notch pathway and wingless transcription. Biol Open [Epub ahead of print]. PubMed ID: 28652317
The Wnt/Wingless (Wg) pathway controls cell fate specification, tissue differentiation and organ development across organisms. Using an in vivo RNAi screen to identify novel kinase and phosphatase regulators of the Wg pathway, subunits of the serine threonine phosphatase Protein phosphatase 4 (PP4) were identifed. Knockdown of the catalytic and the regulatory subunits of PP4 cause reductions in the Wg pathway targets Senseless and Distal-less. PP4 regulates the Wg pathway by controlling Notch-driven wg transcription. Genetic interaction experiments identified that PP4 likely promotes Notch signaling within the nucleus of the Notch-receiving cell. Although the PP4 complex is implicated in various cellular processes, its role in the regulation of Wg and Notch pathways was previously uncharacterized. This study identifies a novel role of PP4 in regulating Notch pathway, resulting in aberrations in Notch-mediated transcriptional regulation of the Wingless ligand. Furthermore, it was shown that PP4 regulates proliferation independent of its interaction with Notch.
Jossin, Y., Lee, M., Klezovitch, O., Kon, E., Cossard, A., Lien, W. H., Fernandez, T. E., Cooper, J. A. and Vasioukhin, V. (2017). Llgl1 connects cell polarity with cell-cell adhesion in embryonic neural stem cells. Dev Cell 41(5): 481-495.e485. PubMed ID: 28552558
Evolutionary Homolog Study
Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. Mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1fl/fl;), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. This study shows that LLGL1 directly binds to and promotes internalization of N-cadherin (see Drosophila N-cadherin), and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells.

Sunday, August 6th

Ballinger, M. J. and Perlman, S. J. (2017). Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila. PLoS Pathog 13(7): e1006431. PubMed ID: 28683136
While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. This study tested the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. Strong evidence was found for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the alpha-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, was found to escape protection by Spiroplasma, and its ribosomes do not show high levels of depurination. It was also shown that fly ribosomes show little evidence of targeting by RIPs. Finally, the genome of D. neotestacea's defensive Spiroplasma was found to encode a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.
Osada, N., Miyagi, R. and Takahashi, A. (2017). Cis- and trans-regulatory effects on gene expression in a natural population of Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 28615283
Cis- and trans-regulatory mutations are important contributors to transcriptome evolution. Quantifying their relative contributions to intraspecific variation in gene expression is essential for understanding the population genetic processes that underlie evolutionary changes in gene expression. This study has examined this issue by quantifying genome-wide allele specific expression (ASE) variation using a crossing scheme that produces F1 hybrids between 18 different Drosophila melanogaster strains sampled from the Drosophila Genetic Reference Panel (DGRP) and a reference strain from another population. Head and body samples from F1 adult females were subjected to RNA-seq and the subsequent ASE quantification. Cis- and trans-regulatory effects on expression variation were estimated from these data. A higher proportion of genes showed significant cis-regulatory variation (~28%) than those showed significant trans-regulatory variation (~9%). The sizes of cis-regulatory effects on expression variation were 1.98 and 1.88 times larger than trans-regulatory effects in heads and bodies, respectively. A generalized linear model analysis revealed that both cis- and trans-regulated expression variation was strongly associated with nonsynonymous nucleotide diversity and tissue specificity. Interestingly, trans-regulated variation showed a negative correlation with local recombination rate. Also, analysis on proximal transposon element (TE) insertions suggested that they affect transcription levels of ovary-expressed genes more pronouncedly than genes not expressed in the ovary, possibly due to defense mechanisms against TE mobility in the germline. Collectively, this detailed quantification of ASE variations from a natural population has revealed a number of new relationships between genomic factors and the effects of cis- and trans-regulatory factors on expression variation.
Gibert, J. M., Blanco, J., Dolezal, M., Nolte, V., Peronnet, F. and Schlotterer, C. (2017). Strong epistatic and additive effects of linked candidate SNPs for Drosophila pigmentation have implications for analysis of genome-wide association studies results. Genome Biol 18(1): 126. PubMed ID: 28673357
The mapping resolution of genome-wide association studies (GWAS) is limited by historic recombination events and effects are often assigned to haplotype blocks rather than individual SNPs. It is not clear how many of the SNPs in the block, and which ones, are causative. Drosophila pigmentation is a powerful model to dissect the genetic basis of intra-specific and inter-specific phenotypic variation. Three tightly linked SNPs in the t-MSE enhancer have been identified in three D. melanogaster populations as major contributors to female abdominal pigmentation. This enhancer controls the expression of the pigmentation gene tan (t) in the abdominal epidermis. Two of the three SNPs were confirmed in an independent study using the D. melanogaster Genetic Reference Panel established from a North American population. This study determined the functional impact of SNP1, SNP2, and SNP3 using transgenic lines to test all possible haplotypes in vivo. All three candidate SNPs contribute to female Drosophila abdominal pigmentation. Interestingly, only two SNPs agree with the effect predicted by GWAS; the third one goes in the opposite direction because of linkage disequilibrium between multiple functional SNPs. The experimental design uncovered strong additive effects for the three SNPs, but significant epistatic effects explaining up to 11% of the total variation. These results suggest that linked causal variants are important for the interpretation of GWAS and functional validation is needed to understand the genetic architecture of traits.
Leung, W., et al. (2017). Retrotransposons are the major contributors to the expansion of the Drosophila ananassae Muller F element. G3 (Bethesda). PubMed ID: 28667019
The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (~5.2 Mb) is the smallest chromosome in Drosophila melanogaster, but it is substantially larger (>18.7 Mb) in Drosophila ananassae. To identify the major contributors to the expansion of the F element and to assess their impact, the genome sequence was improved and the genes in a 1.4 Mb region of the D. ananassae F element, and a 1.7 Mb region from the D element were annotated for comparison. Transposons (particularly LTR and LINE retrotransposons) were found to be major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F element genes exhibit distinct characteristics compared to D element genes (e.g., larger coding spans, larger introns, more coding exons, lower codon bias), but these differences are exaggerated in D. ananassae. Compared to D. melanogaster, the codon bias observed in D. ananassae F element genes can primarily be attributed to mutational biases instead of selection. The 5' ends of F element genes in both species are enriched in H3K4me2 while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves understanding of how transposons can affect genome size and how genes can function within highly repetitive domains.
Morimoto, J., Ponton, F., Tychsen, I., Cassar, J. and Wigby, S. (2017). Interactions between the developmental and adult social environments mediate group dynamics and offspring traits in Drosophila melanogaster. Sci Rep 7(1): 3574. PubMed ID: 28620201
Developmental conditions can strongly influence adult phenotypes and social interactions, which in turn affect key evolutionary processes such as sexual selection and sexual conflict. While the implications of social interactions in phenotypically mixed populations at the individual level are increasingly well known, how these effects influence the fate of groups remains poorly understood, which limits understanding of the broader ecological implications. To address this problem this study manipulated adult phenotypes and social composition in Drosophila melanogaster - by experimentally manipulating the larval density of the group-members - and measured a range of group-level outcomes across the lifespan of groups. Adult groups composed of exclusively low larval-density individuals showed high courtship levels, and low early reproductive rates, group growth rates, offspring mass and offspring eclosion success, relative to high larval-density or mixed larval-density groups. Furthermore, high larval-density groups had lower survival. Offspring mass increased with time, but at a reduced rate in groups when male group members (but not females) were from a mixture of larval-densities; peak reproductive rates were also earlier in these groups. These results suggest that that variation in developmental conditions experienced by adult group members can modify the reproductive output of groups.
de Lima, L. G., Svartman, M. and Kuhn, G. C. S. (2017). Dissecting the satellite DNA landscape in three cactophilic Drosophila sequenced genomes. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 28659292
Eukayote genomes are replete with repetitive DNAs. This class includes tandemly repeated satellite DNAs (satDNA) which are among the most abundant, fast evolving (yet poorly studied) genomic components. This study used high throughput sequencing data from three cactophilic Drosophila species, D. buzzatii, D. seriema and D. mojavensis, to access and study their whole satDNA landscape. Five satDNAs were identified, three previously described (pBuM, DBC-150 and CDSTR198) and two novel ones (CDSTR138 and CDSTR130). Only pBuM is shared among all three species. The satDNA repeat length falls within only two classes, between 130-200bp or between 340-390bp. FISH on metaphase and polytene chromosomes revealed the presence of satDNA arrays in at least one of the following genomic compartments: centromeric, telomeric, subtelomeric or dispersed along euchromatin. The chromosomal distribution ranges from a single chromosome to almost all chromosomes of the complement. Interspersion were revealed between pBuM and CDSTR130 in the microchromosomes of D. mojavensis. Phylogenetic analyses showed that the pBuM satDNA underwent concerted evolution at both interspecific and intraspecific levels. Based on RNAseq data, transcription activity was found for pBuM (in D. mojavensis) and CDSTR198 (in D. buzzatii) in all five analyzed developmental stages, most notably in pupae and adult males. These data revealed that cactophilic Drosophila present the lowest amount of satDNAs (1.9% to 2.9%) within the Drosophila genus reported so far.

Saturday, August 5th

Merk, K., Breinig, M., Bottcher, R., Krebs, S., Blum, H., Boutros, M. and Forstemann, K. (2017). Splicing stimulates siRNA formation at Drosophila DNA double-strand breaks. PLoS Genet 13(6): e1006861. PubMed ID: 28628606
DNA double-strand breaks trigger the production of locus-derived siRNAs. In flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. A genome-wide RNAi-screen was performed in cultured Drosophila cells, that revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation. This observation was validated through site-specific DNA cleavage with CRISPR-cas9 followed by deep sequencing of small RNAs. DNA breaks in intron-less genes or upstream of a gene's first intron did not efficiently trigger siRNA production. When DNA double-strand breaks were induced downstream of an intron, however, this led to robust siRNA generation. Furthermore, a downstream break slowed down splicing of the upstream intron and a detailed analysis of siRNA coverage at the targeted locus revealed that unspliced pre-mRNA contributes the sense strand to the siRNA precursor. Since splicing factors are stimulating the response but unspliced transcripts are entering the siRNA biogenesis, the spliceosome is apparently stalled in a pre-catalytic state and serves as a signaling hub. It is concluded that convergent transcription at DNA breaks is stimulated by a splicing dependent control process. The resulting double-stranded RNA is converted into siRNAs that instruct the degradation of cognate mRNAs. In addition to a potential role in DNA repair, the break-induced transcription may thus be a means to cull improper RNAs from the transcriptome of Drosophila melanogaster. Since the splicing factors identified in the screen also stimulated siRNA production from high copy transgenes, it is possible that this surveillance mechanism serves in genome defense beyond DNA double-strand breaks.
Kunzelmann, S. and Forstemann, K. (2017). Reversible perturbations of gene regulation after genome editing in Drosophila cells. PLoS One 12(6): e0180135. PubMed ID: 28658280
The prokaryotic phage defense CRISPR/cas-system has developed into a versatile toolbox for genome engineering and genetic studies in many organisms. While many efforts were spent on analyzing the consequences of off-target effects, only few studies addressed side-effects that occur due to the targeted manipulation of the genome. This study shows that the CRISPR/cas9-mediated integration of an epitope tag in combination with a selection cassette can trigger an siRNA-mediated, epigenetic genome surveillance pathway in Drosophila melanogaster cells. After homology-directed insertion of the sequence coding for the epitope tag and the selection marker, a moderate level of siRNAs covering the inserted sequence and extending into the targeted locus was detected. This response affected protein levels less than two-fold and it persisted even after single cell cloning. However, removal of the selection cassette abolished the siRNA generation, demonstrating that this response is reversible. Consistently, marker-free genome engineering did not trigger the same surveillance mechanism. These two observations indicate that the selection cassette that was employed induces an aberrant transcriptional arrangement and ultimately sets off the siRNA production. There have been prior concerns about undesirable effects induced by selection markers, but fortunately it was possible to show that at least one of the epigenetic changes reverts as the marker gene is excised. Although the effects observed were rather weak (less than twofold de-repression upon ago2 or dcr-2 knock-down), it is recommended that when selection markers are used during genome editing, a strategy for their subsequent removal should always be included.
Fast, I., Hewel, C., Wester, L., Schumacher, J., Gebert, D., Zischler, H., Berger, C. and Rosenkranz, D. (2017). Temperature-responsive miRNAs in Drosophila orchestrate adaptation to different ambient temperatures. RNA [Epub ahead of print]. PubMed ID: 28630141
The majority of Drosophila genes are expressed in a temperature-dependent manner, but the way in which small RNAs may contribute to this effect is completely unknown, since ideas of how small RNA transcriptomes change as a function of temperature are lacking. Applying high throughput sequencing techniques complemented by quantitative real-time PCR experiments, this study demonstrates that altered ambient temperature induces drastic, but reversible changes in sequence composition and total abundance of both, miRNA- and piRNA populations. Further, mRNA sequencing reveals that the expression of miRNAs and their predicted target transcripts correlates inversely, suggesting that temperature-responsive miRNAs drive adaptation to different ambient temperatures on the transcriptome level. Finally, shifts in temperature were shown to affect both, primary and secondary piRNA pools, and the observed aberrations are consistent with altered expression levels of the involved Piwi-pathway factors. It was further reasoned that enhanced ping-pong processing at 29 ° C is driven by dissolved RNA secondary structures at higher temperatures, uncovering target sites that are not accessible at low temperatures. Together, these results show that small RNAs are an important part of epigenetic regulatory mechanisms that ensure homeostasis and adaptation under fluctuating environmental conditions.
Kristo, I., Bajusz, C., Borsos, B. N., Pankotai, T., Dopie, J., Jankovics, F., Vartiainen, M. K., Erdelyi, M. and Vilmos, P. (2017). The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 28554770
Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. This study shows that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on these results it is proposed that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export.
Hara, M., Petrova, B. and Orr-Weaver, T. L. (2017). Control of PNG kinase, a key regulator of mRNA translation, is coupled to meiosis completion at egg activation. Elife 6. PubMed ID: 28555567
The oocyte-to-embryo transition involves extensive changes in mRNA translation, regulated in Drosophila by the PNG kinase complex whose activity is shown in this study to be under precise developmental control. Despite presence of the catalytic PNG subunit and the PLU and GNU activating subunits in the mature oocyte, GNU is phosphorylated at Cyclin B/CDK1 sites and unable to bind PNG and PLU. In vitro phosphorylation of GNU by CyclinB/CDK1 blocks activation of PNG. Meiotic completion promotes GNU dephosphorylation and PNG kinase activation to regulate translation. The critical regulatory effect of phosphorylation is shown by replacement in the oocyte with a phosphorylation-resistant form of GNU, which promotes PNG-GNU complex formation, elevation of Cyclin B, and meiotic defects consistent with premature PNG activation. After PNG activation GNU is destabilized, thus inactivating PNG. This short-lived burst in kinase activity links development with maternal mRNA translation and ensures irreversibility of the oocyte-to-embryo transition.
Kan, L., Grozhik, A. V., Vedanayagam, J., Patil, D. P., Pang, N., Lim, K. S., Huang, Y. C., Joseph, B., Lin, C. J., Despic, V., Guo, J., Yan, D., Kondo, S., Deng, W. M., Dedon, P. C., Jaffrey, S. R. and Lai, E. C. (2017). The m6A pathway facilitates sex determination in Drosophila. Nat Commun 8: 15737. PubMed ID: 28675155
The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. This study establish the Drosophila system to study the m6A pathway. miCLIP was applied to map m6A across embryogenesis, characterize its m6A 'writer' complex, validate its YTH 'readers' CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, the transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway.

Friday, August 4th

Hidalgo, S., Molina-Mateo, D., Escobedo, P., Zarate, R. V., Fritz, E., Fierro, A., Perez, E. G., Iturriaga-Vasquez, P., Reyes-Parada, M., Varas, R., Fuenzalida-Uribe, N. and Campusano, J. M. (2017). Characterization of a novel Drosophila SERT mutant: insights on the contribution of the serotonin neural system to behaviors. ACS Chem Neurosci [Epub ahead of print]. PubMed ID: 28665105
A better comprehension on how different molecular components of the serotonergic system contribute to the adequate regulation of behaviors in animals is essential in the interpretation on how they are involved in neuropsychiatric and pathological disorders. It is possible to study these components in "simpler" animal models including the fly Drosophila melanogaster, given that most of the components of the serotonergic system are conserved between vertebrates and invertebrates. This study has attempted advance understanding on how the serotonin plasma membrane transporter (SERT) contributes to serotonergic neurotransmission and behaviors in Drosophila. A mutant for Drosophila SERT (dSERT) was characterized, and additionally a highly selective serotonin-releasing drug, 4-methylthioamphetamine (4-MTA), was used whose mechanism of action involves the SERT protein. The results show that dSERT mutant animals exhibit an increased survival rate in stress conditions, increased basal motor behavior and decreased levels in an anxiety-related parameter, centrophobism. It was also shown that 4-MTA increases the negative chemotaxis towards a strong aversive odorant, Benzaldehyde. The neurochemical data suggest that this effect is mediated by dSERT and depends on 4-MTA-increased release of serotonin in the fly brain. The in silico data support the idea that these effects are explained by specific interactions between 4-MTA and dSERT. In sum neurochemical, in-silico and behavioral analyses demonstrate the critical importance of the serotonergic system and particularly dSERT functioning in modulating several behaviors in Drosophila.
Koemans, T. S., Oppitz, C., Donders, R. A. T., van Bokhoven, H., Schenck, A., Keleman, K. and Kramer, J. M. (2017). Drosophila courtship conditioning as a measure of learning and memory. J Vis Exp(124) [Epub ahead of print]. PubMed ID: 28605393
Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male's copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to naive flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized in this study. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.
Hu, C., Petersen, M., Hoyer, N., Spitzweck, B., Tenedini, F., Wang, D., Gruschka, A., Burchardt, L. S., Szpotowicz, E., Schweizer, M., Guntur, A. R., Yang, C. H. and Soba, P. (2017). Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior. Nat Neurosci [Epub ahead of print]. PubMed ID: 28604684
Nociception is an evolutionarily conserved mechanism to encode and process harmful environmental stimuli. Like most animals, Drosophila melanogaster larvae respond to a variety of nociceptive stimuli, including noxious touch and temperature, with stereotyped escape responses through activation of multimodal nociceptors. How behavioral responses to these different modalities are processed and integrated by the downstream network remains poorly understood. By combining trans-synaptic labeling, ultrastructural analysis, calcium imaging, optogenetics and behavioral analyses, this study uncovered a circuit specific for mechanonociception but not thermonociception. Notably, integration of mechanosensory input from innocuous and nociceptive sensory neurons is required for robust mechanonociceptive responses. It was further shown that neurons integrating mechanosensory input facilitate primary nociceptive output by releasing short neuropeptide F, the Drosophila neuropeptide Y homolog. These findings unveil how integration of somatosensory input and neuropeptide-mediated modulation can produce robust modality-specific escape behavior.
Kozlov, A., Jaumouille, E., Machado Almeida, P., Koch, R., Rodriguez, J., Abruzzi, K. C. and Nagoshi, E. (2017). A screening of UNF targets identifies Rnb, a novel regulator of Drosophila circadian rhythms. J Neurosci [Epub ahead of print]. PubMed ID: 28592698
Behavioral circadian rhythms are controlled by multi-oscillator networks comprising functionally different subgroups of clock neurons. Studies have demonstrated that molecular clocks in the fruit fly Drosophila melanogaster are regulated differently in clock neuron subclasses to support their specific functions. The nuclear receptor unfulfilled (unf) represents a regulatory node that provides the small ventral Lateral Neurons (s-LNvs) unique characteristics as the master pacemaker. Previous work has shown that UNF interacts with the s-LNv molecular clocks by regulating transcription of the core clock gene period (per). To gain more insight into the mechanisms by which UNF contributes to the functioning of the circadian master pacemaker, this study identified UNF target genes using chromatin immunoprecipitation. The data demonstrate that a previously uncharacterized gene CG7837, which this study has termed R and B (Rnb), acts downstream of UNF to regulate the function of s-LNvs as the master circadian pacemaker. Mutations and LNv-targeted adult-restricted knockdown of Rnb impair locomotor rhythms. RNB localizes to the nucleus and its loss-of-function blunts the molecular rhythms and output rhythms of the s-LNvs, particularly the circadian rhythms in PDF accumulation and axonal arbor remodeling. These results establish a second pathway by which UNF interacts with the molecular clocks in the s-LNvs and highlight the mechanistic differences in the molecular clockwork within the pacemaker circuit.

Thursday, August 3rd

Jin, P., Bulkley, D., Guo, Y., Zhang, W., Guo, Z., Huynh, W., Wu, S., Meltzer, S., Cheng, T., Jan, L. Y., Jan, Y. N. and Cheng, Y. (2017). Electron cryo-microscopy structure of the mechanotransduction channel NOMPC. Nature 547(7661): 118-122. PubMed ID: 28658211
Mechanosensory transduction for senses such as proprioception, touch, balance, acceleration, hearing and pain relies on mechanotransduction channels, which convert mechanical stimuli into electrical signals in specialized sensory cells. How force gates mechanotransduction channels is a central question in the field, for which there are two major models. One is the membrane-tension model: force applied to the membrane generates a change in membrane tension that is sufficient to gate the channel, as in the bacterial MscL channel and certain eukaryotic potassium channels. The other is the tether model: force is transmitted via a tether to gate the channel. The transient receptor potential (TRP) channel NOMPC is important for mechanosensation-related behaviours such as locomotion, touch and sound sensation across different species including Caenorhabditis elegans, Drosophila and zebrafish. NOMPC is the founding member of the TRPN subfamily, and is thought to be gated by tethering of its ankyrin repeat domain to microtubules of the cytoskeleton. Thus, a goal of studying NOMPC is to reveal the underlying mechanism of force-induced gating, which could serve as a paradigm of the tether model. NOMPC fulfils all the criteria that apply to mechanotransduction channels and has 29 ankyrin repeats, the largest number among TRP channels. A key question is how the long ankyrin repeat domain is organized as a tether that can trigger channel gating. This study presents a de novo atomic structure of Drosophila NOMPC determined by single-particle electron cryo-microscopy. Structural analysis suggests that the ankyrin repeat domain of NOMPC resembles a helical spring, suggesting its role of linking mechanical displacement of the cytoskeleton to the opening of the channel. The NOMPC architecture underscores the basis of translating mechanical force into an electrical signal within a cell.
Greenblatt Ben-El, R. T., Hassan, A. and Salzberg, A. (2017). Loss of thrombospondin reveals a possible role for the extracellular matrix in chordotonal cap cell elongation. Int J Dev Biol 61(3-4-5): 311-318. PubMed ID: 28621428
In the Drosophila larva, major proprioceptive input is provided to the brain by sub-epidermal stretch receptors called chordotonal organs (ChO). Similarly to the body wall muscle that needs to be attached on both of its sides to the larval exoskeleton in order to generate movement, the sensory unit of a ChO must be stably anchored to the cuticle on both of its sides in order to sense the relative displacement of body parts. Through an RNAi screen this study has identified thrombospondin (Tsp), a secreted calcium binding glycoprotein, as a critical component in the anchoring of ChOs to the cuticle. The Tsp protein starts to accumulate in the extracellular matrix (ECM) surrounding the ChO attachment cells towards the end of embryogenesis and that it becomes highly concentrated at the attachment junction during larval stages. In the absence of Tsp, the ChO's accessory cells fail to form a stable junction with their epidermal attachment cells and organ integrity is not maintained. Tsp is a known player in the establishment of the myotendinous junctions in both invertebrates and vertebrates. Thus, these findings extend the known similarities between muscle-attachment and ChO-attachment cells. In addition to its role in establishing the ChO attachment junctions, Tsp was found to affect ligament cell migration and cap cell elongation. Most interestingly, the Tsp protein was found to decorate the ChO cap cells along their entire length, suggesting that the elongated cap cells are supported by the ECM to which they attach via integrin-based, Tsp-dependent, adhesion plaques. The ECM enwrapping the cap cells is probably important for keeping the cap cells fasciculate and may also provide mechanical support that allows the extremely elongated cells to maintain tension.
Gorur-Shandilya, S., Demir, M., Long, J., Clark, D. A. and Emonet, T. (2017). Olfactory receptor neurons use gain control and complementary kinetics to encode intermittent odorant stimuli. Elife 6. PubMed ID: 28653907
Insects find food and mates by navigating odorant plumes that can be highly intermittent, with intensities and durations that vary rapidly over orders of magnitude. Much is known about olfactory responses to pulses and steps, but it remains unclear how olfactory receptor neurons (ORNs) detect the intensity and timing of natural stimuli, where the absence of scale in the signal makes detection a formidable olfactory task. By stimulating Drosophila ORNs in vivo with naturalistic and Gaussian stimuli, this study shows that ORNs adapt to stimulus mean and variance, and that adaptation and saturation contribute to naturalistic sensing. Mean-dependent gain control followed the Weber-Fechner relation and occurred primarily at odor transduction, while variance-dependent gain control occurred at both transduction and spiking. Transduction and spike generation possessed complementary kinetic properties, that together preserved the timing of odorant encounters in ORN spiking, regardless of intensity. Such scale-invariance could be critical during odor plume navigation.
Herzmann, S., Krumkamp, R., Rode, S., Kintrup, C. and Rumpf, S. (2017). PAR-1 promotes microtubule breakdown during dendrite pruning in Drosophila. Embo J 36(13): 1981-1991. PubMed ID: 28554895
Pruning of unspecific neurites is an important mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions. Prior to severing, dendritic microtubules undergo local disassembly, and dendrites thin extensively through local endocytosis. Microtubule disassembly requires a katanin homologue, but the signals initiating microtubule breakdown are not known. This study shows that the kinase PAR-1 is required for pruning and dendritic microtubule breakdown. The data show that neurons lacking PAR-1 fail to break down dendritic microtubules, and PAR-1 is required for an increase in neuronal microtubule dynamics at the onset of metamorphosis. Mammalian PAR-1 is a known Tau kinase, and genetic interactions suggest that PAR-1 promotes microtubule breakdown largely via inhibition of Tau also in Drosophila. Finally, PAR-1 is also required for dendritic thinning, suggesting that microtubule breakdown might precede ensuing plasma membrane alterations. These results shed light on the signaling cascades and epistatic relationships involved in neurite destabilization during dendrite pruning.

Wednesday, August 2nd

Huang, J., Reilein, A. and Kalderon, D. (2017). Yorkie and Hedgehog independently restrict BMP production in Escort cells to permit germline differentiation in the Drosophila ovary. Development. PubMed ID: 28619819
Multiple signaling pathways guide the behavior and differentiation of both germline stem cells (GSCs) and somatic stem cells (FSCs) in the Drosophila germarium, necessitating careful control of signal generation, range and responses. Signal integration involves Escort Cells (ECs), which promote differentiation of the GSC derivatives they envelop, provide niche signals for FSCs and derive directly from FSCs in adults. Hedgehog (Hh) signaling induces the Hippo pathway effector Yorkie (Yki) to promote proliferation and maintenance of FSCs but Hh also signals to ECs, which are quiescent. This study shows that in ECs both Hh and Yki limit production of BMP ligands to allow germline differentiation. Loss of Yki produced a more severe germarial phenotype than loss of Hh signaling and principally induced a different BMP ligand. Moreover, Yki activity reporters and epistasis tests showed that Yki does not mediate the key actions of Hh signaling in ECs. Thus, both the coupling and output of Hh and Yki signaling pathways differ between FSCs and ECs despite their proximity and the fact that FSCs give rise directly to ECs.
Hsu, T. H., Yang, C. Y., Yeh, T. H., Huang, Y. C., Wang, T. W. and Yu, J. Y. (2017). The Hippo pathway acts downstream of the Hedgehog signaling to regulate follicle stem cell maintenance in the Drosophila ovary. Sci Rep 7(1): 4480. PubMed ID: 28667262
The Hippo pathway is conserved and plays important roles in organ size control. The core components of the Hippo pathway are two kinases Hippo (Hpo), Warts (Wts), and a transcription-co-activator Yorkie (Yki). Yki activity is regulated by phosphorylation, which affects its nuclear localization and stability. To determine the role of the Hippo pathway in stem cells, this study examined follicle stem cells (FSCs) in the Drosophila ovary. Yki is detected in the nucleus of FSCs. Knockdown of yki in the follicle cell lineage leads to a disruption of the follicular epithelium. Mitotic clones of FSCs mutant for hpo or wts are maintained in the niche and tend to replace the other FSCs, and FSCs mutant for yki are rapidly lost, demonstrating that the Hippo pathway is both required and sufficient for FSC maintenance. Using genetic interaction analyses, the Hedgehog pathway was demonstrated to act upstream of the Hippo pathway in regulating FSC maintenance. The nuclear localization of Yki is enhanced when the Hedgehog signaling is activated. Furthermore, a constitutively active but not a wild-type Yki promotes FSC maintenance as activation of the Hedgehog signaling does, suggesting that the Hedgehog pathway regulates Yki through a post-translational mechanism in maintaining FSCs.
Inaba, M., Sorenson, D. R., Kortus, M., Salzmann, V. and Yamashita, Y. M. (2017). Merlin is required for coordinating proliferation of two stem cell lineages in the Drosophila testis. Sci Rep 7(1): 2502. PubMed ID: 28566755
Although the mechanisms that balance self-renewal and differentiation of a stem cell lineage have been extensively studied, it remains poorly understood how tissues that contain multiple stem cell lineages maintain balanced proliferation among distinct lineages: when stem cells of a particular lineage proliferate, how do the other lineages respond to maintain the correct ratio of cells among linages? This study shows that Merlin (Mer), a homolog of the human tumor suppressor neurofibromatosis 2, is required to coordinate proliferation of germline stem cells (GSCs) and somatic cyst stem cells (CySCs) in the Drosophila testis. Mer mutant CySCs fail to coordinate their proliferation with that of GSCs in multiple settings, and can be triggered to undergo tumorous overproliferation. Mer executes its function by stabilizing adherens junctions. Given the known role of Mer in contact-dependent inhibition of proliferation, it is proposed that the proliferation of CySCs are regulated by crowdedness, or confluency, of cells in their lineage with respect to that of germline, thereby coordinating the proliferation of two lineages.
Hamada-Kawaguchi, N. and Yamamoto, D. (2017). Ovarian polarity and cell shape determination by Btk29A in Drosophila. Genesis [Epub ahead of print]. PubMed ID: 28639397
Drosophila Btk29A is a Tec family nonreceptor tyrosine kinase, the ortholog of which causes X-linked agammagluburinemia in humans when mutant. In Btk29AficP mutant ovaries, multiple defects are observed: extra polar cells form ectopically; osk mRNA fails to accumulate posteriorly in mature oocytes; the shape and alignment of follicle cells are grossly distorted. All these phenotypes are rescued by selectively overexpressing the type 2 isoform of wild-type Btk29A in follicle cells. Expression of certain proteins enriched in adherens junctions is markedly affected in Btk29AficP mutants; the anterior-posterior gradient normally observed in the expression of DE-Cadherin and Armadillo are lost and Canoe is sequestered from adherens junctions. Intriguingly, tyrosine phosphorylation of Canoe is reduced in Btk29AficP mutants. It is proposed that Btk29A is required for the establishment of egg chamber polarity presumably through the regulation of subcellular localization of its downstream proteins, including Cno.

Tuesday, August 1st

Davis, T. L. and Rebay, I. (2017). Antagonistic regulation of the second mitotic wave by Eyes absent-Sine oculis and Combgap coordinates proliferation and specification in the Drosophila retina. Development. PubMed ID: 28619818
The transition from proliferation to specification is fundamental to the development of appropriately patterned tissues. In the developing Drosophila eye, Eyes absent (Eya) and Sine oculis (So) orchestrate the progression of progenitor cells from asynchronous cell division to G1 arrest and neuronal specification at the morphogenetic furrow. This study uncovered a novel role for Eya and So in promoting cell cycle exit in the Second Mitotic Wave (SMW), a synchronized, terminal cell division that occurs several hours after passage of the furrow. Combgap (Cg), a zinc-finger transcription factor, antagonizes Eya-So function in the SMW. Based on Cg's ability to attenuate Eya-So transcriptional output in vivo and in cultured cells and on meta-analysis of their chromatin occupancy profiles, it is speculated that Cg limits Eya-So activation of select target genes posterior to the furrow to ensure properly timed mitotic exit. This work supports a model in which context-specific modulation of transcriptional activity enables Eya and So to promote both entry into and exit from the cell cycle in a distinct spatiotemporal sequence.
Dahal, G. R., Pradhan, S. J. and Bates, E. A. (2017). Inwardly rectifying potassium channels regulate Dpp release in the Drosophila wing disc. Development [Epub ahead of print]. PubMed ID: 28684627
Loss of embryonic ion channel function leads to morphological defects, but the underlying reason for these defects remains elusive. This study shows that inwardly-rectifying potassium (Irk) channels regulate release of a Drosophila bone morphogenetic protein (BMP/Dpp) in the developing fly wing and this is necessary for developmental signaling. Inhibition of Irk channels decreases the incidence of distinct Dpp-GFP release events above baseline fluorescence while leading to broader distribution of Dpp-GFP. Work by others in different cell types show Irk channels regulate peptide release by modulating membrane potential and calcium levels. This study found calcium transients in the developing wing and inhibition of Irk channels reduces the duration and amplitude of calcium transients. Depolarization with high extracellular potassium evokes Dpp release. Taken together, these data implicate Irk channels as a requirement for regulated release of Dpp, highlighting the importance of the temporal pattern of Dpp presentation for morphogenesis of the wing.
Barrio, L. and Milan, M. (2017). Boundary Dpp promotes growth of medial and lateral regions of the Drosophila wing. Elife 6. PubMed ID: 28675372
The gradient of Decapentaplegic (Dpp) in the Drosophila wing has served as a paradigm to characterize the role of morphogens in regulating patterning. However, the role of this gradient in regulating tissue size is a topic of intense debate as proliferative growth is homogenous. This study combined the Gal4/UAS system and a temperature-sensitive Gal80 molecule to induce RNAi-mediated depletion of dpp and characterise the spatial and temporal requirement of Dpp in promoting growth. Dpp emanating from the AP compartment boundary was shown to be required throughout development to promote growth by regulating cell proliferation and tissue size. Dpp regulates growth and proliferation rates equally in central and lateral regions of the developing wing appendage and reduced levels of Dpp affects similarly the width and length of the resulting wing. Evidence is presented supporting the proposal that graded activity of Dpp is not an absolute requirement for wing growth.
Heinze, S. D., Kohlbrenner, T., Ippolito, D., Meccariello, A., Burger, A., Mosimann, C., Saccone, G. and Bopp, D. (2017). CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus. Sci Rep 7(1): 4582. PubMed ID: 28676649
The classic brown body (bwb) mutation in the housefly Musca domestica impairs normal melanization of the adult cuticle. In Drosophila melanogaster, a reminiscent pigmentation defect results from mutations in the yellow gene encoding dopachrome conversion enzyme (DCE). This study demonstrates that the bwb locus structurally and functionally represents the yellow ortholog of Musca domestica, MdY. In bwb Musca strains, two mutant MdY alleles were identified that contain lesions predicted to result in premature truncation of the MdY open reading frame. Wildtype MdY was targeted by CRISPR-Cas9 RNPs and new mutant alleles were generated alleles that fail to complement existing MdY alleles, genetically confirming that MdY is the bwb locus. Further evidence was found for Cas9-mediated interchromosomal recombination between wildtype and mutant bwb alleles. This work resolves the molecular identity of the classic bwb mutation in Musca domestica and establishes the feasibility of Cas9-mediated genome editing in the Musca model
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