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


Wednesday, November 30th, 2016

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Banerjee, S., Venkatesan, A. and Bhat, M. A. (2016). Neurexin, neuroligin and wishful thinking coordinate synaptic cytoarchitecture and growth at neuromuscular junctions. Mol Cell Neurosci [Epub ahead of print]. PubMed ID: 27838296
Using full length and truncated forms of Neurexin (Dnrx) and Neuroligins (Dnlg) together with cell biological analyses and genetic interactions, this study reports novel functions of Dnrx and Dnlg1 in clustering of pre- and postsynaptic proteins, coordination of synaptic growth and ultrastructural organization. Dnrx and Dnlg1 extracellular and intracellular regions are required for proper synaptic growth and localization of Dnlg1 and Dnrx, respectively. dnrx and dnlg1 single and double mutants display altered subcellular distribution of Discs large (Dlg), which is the homolog of mammalian post-synaptic density protein, PSD95. dnrx and dnlg1 mutants also display ultrastructural defects ranging from abnormal active zones, misformed pre- and post-synaptic areas with underdeveloped subsynaptic reticulum. Interestingly, dnrx and dnlg1 mutants have reduced levels of the BMP receptor Wishful thinking (Wit), and Dnrx and Dnlg1 are required for proper localization and stability of Wit. In addition, the synaptic overgrowth phenotype resulting from the overexpression of Dnrx fails to manifest in wit mutants. Phenotypic analyses of dnrx/wit and dnlg1/wit mutants indicate that Dnrx/Dnlg1/Wit coordinate synaptic growth and architecture at the NMJ. These findings also demonstrate that loss of Dnrx and Dnlg1 leads to decreased levels of the BMP co-receptor, Thickveins and the downstream effector phosphorylated Mad at the NMJ synapses indicating that Dnrx/Dnlg1 regulate components of the BMP signaling pathway. Together these findings reveal that Dnrx/Dnlg are at the core of a highly orchestrated process that combines adhesive and signaling mechanisms to ensure proper synaptic organization and growth during NMJ development.
Krill, J. L. and Dawson-Scully, K. (2016). cGMP-dependent protein kinase inhibition extends the upper temperature limit of stimulus-evoked calcium responses in motoneuronal boutons of Drosophila melanogaster larvae. PLoS One 11: e0164114. PubMed ID: 27711243
While the mammalian brain functions within a very narrow range of oxygen concentrations and temperatures, the fruit fly, Drosophila melanogaster, has employed strategies to deal with a much wider range of acute environmental stressors. The foraging (for) gene encodes the cGMP-dependent protein kinase (PKG), has been shown to regulate thermotolerance in many stress-adapted species, including Drosophila, and could be a potential therapeutic target in the treatment of hyperthermia in mammals. Whereas previous thermotolerance studies have looked at the effects of PKG variation on Drosophila behavior or excitatory postsynaptic potentials at the neuromuscular junction (NMJ), little is known about PKG effects on presynaptic mechanisms. This study characterized presynaptic calcium ([Ca2+]i) dynamics at the Drosophila larval NMJ to determine the effects of high temperature stress on synaptic transmission. The neuroprotective role of PKG modulation was investigated both genetically using RNA interference (RNAi), and pharmacologically, to determine if and how PKG affects presynaptic [Ca2+]i dynamics during hyperthermia. PKG activity was found to modulate presynaptic neuronal Ca2+ responses during acute hyperthermia, where PKG activation makes neurons more sensitive to temperature-induced failure of Ca2+ flux and PKG inhibition confers thermotolerance and maintains normal Ca2+ dynamics under the same conditions. Targeted motoneuronal knockdown of PKG using RNAi demonstrated that decreased PKG expression was sufficient to confer thermoprotection. These results demonstrate that the PKG pathway regulates presynaptic motoneuronal Ca2+ signaling to influence thermotolerance of presynaptic function during acute hyperthermia.
Wang, T., Martin, S., Nguyen, T. H., Harper, C. B., Gormal, R. S., Martinez-Marmol, R., Karunanithi, S., Coulson, E. J., Glass, N. R., Cooper-White, J. J., van Swinderen, B. and Meunier, F. A. (2016). Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB. Nat Commun 7: 12976. PubMed ID: 27687129
Axonal retrograde transport of signalling endosomes from the nerve terminal to the soma underpins survival. As each signalling endosome carries a quantal amount of activated receptors, it was hypothesized that it is the frequency of endosomes reaching the soma that determines the scale of the trophic signal. This study shows that upregulating synaptic activity markedly increased the flux of plasma membrane-derived retrograde endosomes (labelled using cholera toxin subunit-B: CTB) in hippocampal neurons cultured in microfluidic devices, and live Drosophila larval motor neurons. Electron and super-resolution microscopy analyses revealed that the fast-moving sub-diffraction-limited CTB carriers contained the TrkB neurotrophin receptor (see Drosophila Ror), transiently activated by synaptic activity in a BDNF-independent manner. Pharmacological and genetic inhibition of TrkB activation selectively prevented the coupling between synaptic activity and the retrograde flux of signalling endosomes. TrkB activity therefore controls the encoding of synaptic activity experienced by nerve terminals, digitalized as the flux of retrogradely transported signalling endosomes.
Jacomin, A. C., Fauvarque, M. O. and Taillebourg, E. (2016). A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila. BMC Cell Biol 17: 36. PubMed ID: 27852225
Lysosomes are the major catabolic compartment within eukaryotic cells, and their biogenesis requires the integration of the biosynthetic and endosomal pathways. Endocytosis and autophagy are the primary inputs of the lysosomal degradation pathway. Endocytosis is specifically needed for the degradation of membrane proteins whereas autophagy is responsible for the degradation of cytoplasmic components. The deubiquitinating enzyme UBPY/USP8 has been identified as being necessary for lysosomal biogenesis and productive autophagy in Drosophila. Because UBPY/USP8 has been widely described for its function in the endosomal system, it was hypothesized that disrupting the endosomal pathway itself may affect the biogenesis of the lysosomes. This study blocked the progression of the endosomal pathway at different levels of maturation of the endosomes by expressing in fat body cells either dsRNAs or dominant negative mutants targeting components of the endosomal machinery: Shibire, Rab4, Rab5, Chmp1 and Rab7. Inhibition of endosomal trafficking at different steps in vivo was observed to be systematically associated with defects in lysosome biogenesis, resulting in autophagy flux blockade. These results show that the integrity of the endosomal system is required for lysosome biogenesis and productive autophagy in vivo.

Tuesday, November 29th

Gruntenko, N. E., Adonyeva, N. V., Burdina, E. V., Karpova, E. K., Andreenkova, O. V., Gladkikh, D. V., Ilinsky, Y. Y. and Rauschenbach, I. Y. (2016). The impact of FOXO on dopamine and octopamine metabolism in Drosophila under normal and heat stress conditions. Biol Open [Epub ahead of print]. PubMed ID: 27754851
The Forkhead BoxO transcription factor (FOXO) is a component of the insulin signalling pathway and plays a role in responding to adverse conditions, such as oxidative stress and starvation. In stressful conditions, FOXO moves from the cytosol to the nucleus where it activates gene expression programmes. This study shows that FOXO in Drosophila melanogaster responds to heat stress as it does to other stressors. The catecholamine signalling pathway is another component of the stress response. In Drosophila, dopamine and octopamine levels rise steeply under heat, nutrition and mechanical stresses, which is followed by a decrease in the activity of synthesis enzymes. This study demonstrates that the nearly twofold decline of FOXO expression in foxoBG01018 mutants results in dramatic changes in the metabolism of dopamine and octopamine and the overall response to stress. The absence of FOXO increases tyrosine decarboxylase activity, the first enzyme in octopamine synthesis, and decreases the enzymatic activity of enzymes in dopamine synthesis, alkaline phosphatase and tyrosine hydroxylase, in young Drosophila females. The juvenile hormone was identified as a mediator of FOXO regulation of catecholamine metabolism. These findings suggest that FOXO is a possible trigger for endocrinological stress reactions.
Gui, J., Huang, Y. and Shimmi, O. (2016). Scribbled optimizes BMP signaling through its receptor internalization to the Rab5 endosome and promote robust epithelial morphogenesis. PLoS Genet 12: e1006424. PubMed ID: 27814354
Epithelial cells are characterized by apical-basal polarity. Intrinsic factors underlying apical-basal polarity are crucial for tissue homeostasis and have often been identified to be tumor suppressors. Patterning and differentiation of epithelia are key processes of epithelial morphogenesis and are frequently regulated by highly conserved extrinsic factors. However, due to the complexity of morphogenesis, the mechanisms of precise interpretation of signal transduction as well as spatiotemporal control of extrinsic cues during dynamic morphogenesis remain poorly understood. Wing posterior crossvein (PCV) formation in Drosophila serves as a unique model to address how epithelial morphogenesis is regulated by secreted growth factors. Decapentaplegic (Dpp), a conserved bone morphogenetic protein (BMP)-type ligand, is directionally trafficked from longitudinal veins (LVs) into the PCV region for patterning and differentiation. These data reveal that the basolateral determinant Scribbled (Scrib) is required for PCV formation through optimizing BMP signaling. Scrib regulates BMP-type I receptor Thickveins (Tkv) localization at the basolateral region of PCV cells and subsequently facilitates Tkv internalization to Rab5 endosomes, where Tkv is active. BMP signaling also up-regulates scrib transcription in the pupal wing to form a positive feedback loop. These data reveal a unique mechanism in which intrinsic polarity genes and extrinsic cues are coupled to promote robust morphogenesis.
Misra, J.R. and Irvine, K.D. (2016). Vamana couples fat signaling to the Hippo pathway. Dev Cell 39(2):254-266. PubMed ID: 27746048
The protocadherins Dachsous and Fat initiate a signaling pathway that controls growth and planar cell polarity by regulating the membrane localization of the atypical myosin Dachs. How Dachs is regulated by Fat signaling has remained unclear. This study identified the vamana gene as playing a crucial role in regulating membrane localization of Dachs and in linking Fat and Dachsous to Dachs regulation. Vamana, an SH3-domain-containing protein, physically associates with and co-localizes with Dachs and promotes its membrane localization. Vamana also associates with the Dachsous intracellular domain and with a region of the Fat intracellular domain that is essential for controlling Hippo signaling and levels of Dachs. Epistasis experiments, structure-function analysis, and physical interaction experiments argue that Fat negatively regulates Dachs in a Vamana-dependent process. These findings establish Vamana as a crucial component of the Dachsous-Fat pathway that transmits Fat signaling by regulating Dachs.
Kubrak, O. I., Lushchak, O. V., Zandawala, M. and Nassel, D. R. (2016). Systemic corazonin signalling modulates stress responses and metabolism in Drosophila. Open Biol 6. PubMed ID: 27810969
Stress triggers cellular and systemic reactions in organisms to restore homeostasis. Mammalian gonadotropin-releasing hormone (GnRH) and its insect orthologue, adipokinetic hormone (AKH), are known for their roles in modulating stress-related behaviour. This study shows that corazonin (Crz), a peptide homologous to AKH/GnRH, also alters stress physiology in Drosophila. The Crz receptor (CrzR) is expressed in salivary glands and adipocytes of the liver-like fat body, and CrzR knockdown targeted simultaneously to both these tissues increases the fly's resistance to starvation, desiccation and oxidative stress, reduces feeding, alters expression of transcripts of Drosophila insulin-like peptides (DILPs), and affects gene expression in the fat body. Furthermore, in starved flies, CrzR-knockdown increases circulating and stored carbohydrates. Thus, these findings indicate that elevated systemic Crz signalling during stress coordinates increased food intake and diminished energy stores to regain metabolic homeostasis. This study suggests that an ancient stress-peptide in Urbilateria evolved to give rise to present-day GnRH, AKH and Crz signalling systems.

Zhang B, Tsai PC, Gonzalez-Celeiro M, Chung O, Boumard B, Perdigoto CN, Ezhkova E, Hsu YC. (2016). Hair follicles' transit-amplifying cells govern concurrent dermal adipocyte production through Sonic Hedgehog. Genes Dev. [Epub ahead of print]. PubMed ID: 27807033
Evolutionary Homolog Study
Growth and regeneration of one tissue within an organ compels accommodative changes in the surrounding tissues. However, the molecular nature and operating logic governing these concurrent changes remain poorly defined. The dermal adipose layer expands concomitantly with hair follicle downgrowth, providing a paradigm for studying coordinated changes of surrounding lineages with a regenerating tissue. This study discovered that hair follicle transit-amplifying cells (HF-TACs) play an essential role in orchestrating dermal adipogenesis through secreting Sonic Hedgehog (SHH; see Drosophila Hedgehog). Depletion of Shh from HF-TACs abrogates both dermal adipogenesis and hair follicle growth. Using cell type-specific deletion of Smo (see Drosophila Smoothened), a gene required in SHH-receiving cells, it was found that SHH does not act on hair follicles, adipocytes, endothelial cells, and hematopoietic cells for adipogenesis. Instead, SHH acts directly on adipocyte precursors, promoting their proliferation and their expression of a key adipogenic gene, peroxisome proliferator-activated receptor γ (Pparg; see Drosophila Eip75b), to induce dermal adipogenesis. This study therefore uncovers a critical role for TACs in orchestrating the generation of both their own progeny and a neighboring lineage to achieve concomitant tissue production across lineages.
Porazinski S, de Navascués J, Yako Y, Hill W, Jones MR, Maddison R, Fujita Y, Hogan C (2016). EphA2 drives the segregation of Ras-transformed epithelial cells from normal neighbors. Curr Biol [Epub ahead of print]. PubMed ID: 27839970
Evolutionary Homolog Study
In epithelial tissues, cells expressing oncogenic Ras (hereafter RasV12 cells; see Drosophila Ras oncogene at 85D) are detected by normal neighbors and as a result are often extruded from the tissue. RasV12 cells are eliminated apically, suggesting that extrusion may be a tumor-suppressive process. Extrusion depends on E-cadherin-based cell-cell adhesions and signaling to the actin-myosin cytoskeleton. However, the signals underlying detection of the RasV12 cell and triggering extrusion are poorly understood. This study identified differential EphA2 (see Drosophila Eph) signaling as the mechanism by which RasV12 cells are detected in epithelial cell sheets. Cell-cell interactions between normal cells and RasV12 cells trigger ephrin-A-EphA2 signaling, which induces a cell repulsion response in RasV12 cells. Concomitantly, RasV12 cell contractility increases in an EphA2-dependent manner. Together, these responses drive the separation of RasV12 cells from normal cells. In the absence of ephrin-A-EphA2 signals, RasV12 cells integrate with normal cells and adopt a pro-invasive morphology. Drosophila Eph (DEph) is detected in segregating clones of RasV12 cells and is functionally required to drive segregation of RasV12 cells in vivo, suggesting that the in vitro findings are conserved in evolution. It is proposed that expression of RasV12 in single or small clusters of cells within a healthy epithelium creates ectopic EphA2 boundaries, which drive the segregation and elimination of the transformed cell from the tissue. Thus, deregulation of Eph/ephrin would allow RasV12 cells to go undetected and expand within an epithelium.

Monday, November 28th

Ma, Z., Stork, T., Bergles, D.E. and Freeman, M.R. (2016). Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour. Nature 539(7629):428-432. PubMed ID: 27828941
Astrocytes associate with synapses throughout the brain and express receptors for neurotransmitters that can increase intracellular calcium (Ca2+). Astrocytic Ca2+ signalling has been proposed to modulate neural circuit activity, but the pathways that regulate these events are poorly defined and in vivo evidence linking changes in astrocyte Ca2+ levels to alterations in neurotransmission or behaviour is limited. This study shows that Drosophila astrocytes exhibit activity-regulated Ca2+ signalling in vivo. Tyramine and octopamine released from neurons expressing tyrosine decarboxylase 2 (Tdc2) signal directly to astrocytes to stimulate Ca2+ increases through the octopamine/tyramine receptor (Oct-TyrR) and the transient receptor potential (TRP) channel Water witch (Wtrw), and astrocytes in turn modulate downstream dopaminergic neurons. Application of tyramine or octopamine to live preparations silenced dopaminergic neurons and this inhibition required astrocytic Oct-TyrR and Wtrw. Increasing astrocyte Ca2+ signalling was sufficient to silence dopaminergic neuron activity, which was mediated by astrocyte endocytic function and adenosine receptors. Selective disruption of Oct-TyrR or Wtrw expression in astrocytes blocked astrocytic Ca2+ signalling and profoundly altered olfactory-driven chemotaxis and touch-induced startle responses. This work identifies Oct-TyrR and Wtrw as key components of the astrocytic Ca2+ signalling machinery, provides direct evidence that octopamine- and tyramine-based neuromodulation can be mediated by astrocytes, and demonstrates that astrocytes are essential for multiple sensory-driven behaviours in Drosophila (Ma, 2016).
Goda, T., Tang, X., Umezaki, Y., Chu, M. L. and Hamada, F. N. (2016). Drosophila DH31 neuropeptide and PDF receptor regulate night-onset temperature preference. J Neurosci 36: 11739-11754. PubMed ID: 27852781
Body temperature exhibits rhythmic fluctuations over a 24 h period and decreases during the night, which is associated with sleep initiation. However, the underlying mechanism of this temperature decrease is largely unknown. Previous work has shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset. Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature, suggesting that their TPR generates their body temperature rhythm. This study demonstrates that the neuropeptide diuretic hormone 31 (DH31) and pigment-dispersing factor receptor (PDFR) contribute to regulate the preferred temperature decrease at night-onset. PDFR and tethered-DH31 expression in dorsal neurons 2 (DN2s) restore the preferred temperature decrease at night-onset, suggesting that DH31 acts on PDFR in DN2s. Notably, it was previously shown that the molecular clock in DN2s is important for TPR. Although PDF (another ligand of PDFR) is a critical factor for locomotor activity rhythms, Pdf mutants exhibit normal preferred temperature decreases at night-onset. This suggests that DH31-PDFR signaling specifically regulates a preferred temperature decrease at night-onset. Thus, it is proposed that night-onset TPR and locomotor activity rhythms are differentially controlled not only by clock neurons but also by neuropeptide signaling in the brain.
Larter, N. K., Sun, J. S. and Carlson, J. R. (2016). Organization and function of Drosophila odorant binding proteins. Elife 5. PubMed ID: 27845621
Odorant binding proteins (Obps) are remarkable in their number, diversity, and abundance, yet their role in olfactory coding remains unclear. They are widely believed to be required for transporting hydrophobic odorants through an aqueous lymph to odorant receptors. This study constructed a map of the Drosophila antenna, in which the abundant Obps are mapped to olfactory sensilla with defined functions. The results lay a foundation for an incisive analysis of Obp function. The map identifies a sensillum type that contains a single abundant Obp, Obp28a. Surprisingly, deletion of the sole abundant Obp in these sensilla does not reduce the magnitude of their olfactory responses. The results suggest that this Obp is not required for odorant transport and that this sensillum does not require an abundant Obp. The results further suggest a novel role for this Obp in buffering changes in the odor environment, perhaps providing a molecular form of gain control.
Huang, T. H., Velho, T. and Lois, C. (2016). Monitoring cell-cell contacts in vivo in transgenic animals. Development 143: 4073-4084. PubMed ID: 27660327
This study used a synthetic genetic system based on ligand-induced intramembrane proteolysis to monitor cell-cell contacts in animals. Upon ligand-receptor interaction in sites of cell-cell contact, the transmembrane domain of an engineered receptor is cleaved by intramembrane proteolysis and releases a protein fragment that regulates transcription in the interacting partners. The system can be used to regulate gene expression between interacting cells, both in vitro and in vivo, in transgenic Drosophila. The system allows for detection of interactions between neurons and glia in the Drosophila nervous system. In addition, it was observed that when the ligand is expressed in subsets of neurons with a restricted localization in the brain it leads to activation of transcription in a selected set of glial cells that interact with those neurons. This system will be useful to monitor cell-cell interactions in animals, and can be used to genetically manipulate cells that interact with one another.

Sunday, November 27th

Laws, K. M. and Drummond-Barbosa, D. (2016). AMP-activated protein kinase has diet-dependent and -independent roles in Drosophila oogenesis. Dev Biol 420(1):90-99. PubMed ID: 27729213
Multiple aspects of organismal physiology influence the number and activity of stem cells and their progeny, including nutritional status. Previous studies demonstrated that Drosophila germline stem cells (GSCs), follicle stem cells (FSCs), and their progeny sense and respond to diet via complex mechanisms involving many systemic and local signals. AMP-activated protein kinase, or AMPK, is a highly conserved regulator of energy homeostasis known to be activated under low cellular energy conditions; however, its role in the ovarian response to diet has not been investigated. This study describes nutrient-dependent and -independent requirements for AMPK in Drosophila oogenesis. AMPK was found to be cell autonomously required for the slow down in GSC and follicle cell proliferation that occurs on a poor diet. Similarly, AMPK activity is necessary in the germline for the degeneration of vitellogenic stages in response to nutrient deprivation. In contrast, AMPK activity is not required within the germline to modulate its growth. Instead, AMPK acts in follicle cells to negatively regulate their growth and proliferation, thereby indirectly limiting the size of the underlying germline cyst within developing follicles. Paradoxically, AMPK is required for GSC maintenance in well-fed flies (when AMPK activity is presumably at its lowest), suggesting potentially important roles for basal AMPK activity in specific cell types. Finally, a nutrient-independent, developmental role was identified for AMPK in cyst encapsulation by follicle cells. These results uncover specific AMPK requirements in multiple cell types in the ovary and suggest that AMPK can function outside of its canonical nutrient-sensing role in specific developmental contexts.
Alexander, J. L., Beagan, K., Orr-Weaver, T. L. and McVey, M. (2016). Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27849606
Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. Drosophila follicle cell developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. This system was used to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. A null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase theta/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants, mus308 displays normal origin firing but reduced fork progression at two regions of rereplication. MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, fork progression is enhanced in the absence of both Drosophila Rad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, these findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context.
Velentzas, A. D., et al. (2016). Targeted downregulation of s36 protein unearths its cardinal role in chorion biogenesis and architecture during Drosophila melanogaster oogenesis. Sci Rep 6: 35511. PubMed ID: 27752139
Drosophila chorion represents a model biological system for the in vivo study of gene activity, epithelial development, extracellular-matrix assembly and morphogenetic-patterning control. It is produced during the late stages of oogenesis by epithelial follicle cells and develops into a highly organized multi-layered structure that exhibits regional specialization and radial complexity. Among the six major proteins involved in chorion's formation, the s36 and s38 ones are synthesized first and regulated in a cell type-specific and developmental stage-dependent manner. In this study, an RNAi-mediated silencing of s36 chorionic-gene expression specifically in the follicle-cell compartment of Drosophila ovary unearths the essential, and far from redundant, role of s36 protein in patterning establishment of chorion's regional specialization and radial complexity. Without perturbing the developmental courses of follicle- and nurse-cell clusters, the absence of s36 not only promotes chorion's fragility but also induces severe structural irregularities on chorion's surface and entirely impairs fly's fertility. Moreover, s36 chorionic protein regulates the number and morphogenetic integrity of dorsal appendages in follicles sporadically undergoing aged fly-dependent stress.
Kimura, S. and Loppin, B. (2016). The Drosophila chromosomal protein Mst77F is processed to generate an essential component of mature sperm chromatin. Open Biol 6. PubMed ID: 27810970
In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function in vivo. Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro, but its role during spermiogenesis remains unclear. This study shows that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35a/Mst35b. Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals.

Saturday, November 26th

Widmann, A., Artinger, M., Biesinger, L., Boepple, K., Peters, C., Schlechter, J., Selcho, M. and Thum, A. S. (2016). Genetic dissection of aversive associative olfactory learning and memory in Drosophila larvae. PLoS Genet 12: e1006378. PubMed ID: 27768692
Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes-besides other forms-a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. This study deconstructed the larval mnemonic organization after aversive olfactory conditioning. After odor-high salt conditioning (establishing an aversive olfactory memory) larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3'5'-monophosphate (cAMP) signaling and synapsin gene function. In addition, this study shows for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution.
Qiu, S., Xiao, C. and Robertson, R. M. (2016). Pulsed light stimulation increases boundary preference and periodicity of episodic motor activity in Drosophila melanogaster. PLoS One 11: e0163976. PubMed ID: 27684063
There is considerable interest in the therapeutic benefits of long-term sensory stimulation for improving cognitive abilities and motor performance of stroke patients. In this study, the effect of chronic sensory stimulation (pulsed light stimulation) on motor activity in w1118 flies was investigated. Flies were exposed to a chronic pulsed light stimulation protocol prior to testing their performance in a standard locomotion assay. Flies responded to pulsed light stimulation with increased boundary preference and travel distance in a circular arena. In addition, pulsed light stimulation increased the power of extracellular electrical activity, leading to the enhancement of periodic electrical activity which was associated with a centrally-generated motor pattern (struggling behavior). In contrast, such periodic events were largely missing in w1118 flies without pulsed light treatment. These data suggest that the sensory stimulation induced a response in motor activity associated with the modifications of electrical activity in the central nervous system (CNS). Finally, without pulsed light treatment, the wild-type genetic background was associated with the occurrence of the periodic activity in wild-type Canton S (CS) flies, and w+ modulated the consistency of periodicity. It is concluded that pulsed light stimulation modifies behavioral and electrophysiological activities in w1118 flies. These data provide a foundation for future research on the genetic mechanisms of neural plasticity underlying such behavioral modification.
Hirano, Y., Ihara, K., Masuda, T., Yamamoto, T., Iwata, I., Takahashi, A., Awata, H., Nakamura, N., Takakura, M., Suzuki, Y., Horiuchi, J., Okuno, H. and Saitoe, M. (2016). Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies. Nat Commun 7: 13471. PubMed ID: 27841260
Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). This study characterized global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Beadex, also know as LIM-only. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and this study identified different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, it was found that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. These results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs.
Garlapow, M. E., et al. (2016). Genetic and genomic response to selection for food consumption in Drosophila melanogaster. Behav Genet [Epub ahead of print]. PubMed ID: 27704301
Food consumption is an essential component of animal fitness; however, excessive food intake in humans increases risk for many diseases. The roles of neuroendocrine feedback loops, food sensing modalities, and physiological state in regulating food intake are well understood, but not the genetic basis underlying variation in food consumption. This study applied ten generations of artificial selection for high and low food consumption in replicate populations of Drosophila melanogaster. The phenotypic response to selection was highly asymmetric, with significant responses only for increased food consumption and minimal correlated responses in body mass and composition. This study assessed the molecular correlates of selection responses by DNA and RNA sequencing of the selection lines. The high and low selection lines had variants with significantly divergent allele frequencies within or near 2081 genes and 3526 differentially expressed genes in one or both sexes. A total of 519 genes were both genetically divergent and differentially expressed between the divergent selection lines. Functional analyses were performed of the effects of RNAi suppression of gene expression and induced mutations for 27 of these candidate genes that have human orthologs and the strongest statistical support, and it was confirmed that 25 (93 %) affected the mean and/or variance of food consumption.
Wystrach, A., Lagogiannis, K. and Webb, B. (2016). Continuous lateral oscillations as a core mechanism for taxis in Drosophila larvae. Elife 5. PubMed ID: 27751233
Taxis behaviour, chemotaxis for example, in Drosophila larva is thought to consist of distinct control mechanisms triggering specific actions. This study supports a simpler hypothesis: that taxis results from direct sensory modulation of continuous lateral oscillations of the anterior body, sparing the need for 'action selection'. Analysis of larvae motion reveals a rhythmic, continuous lateral oscillation of the anterior body, encompassing all head-sweeps, small or large, without breaking the oscillatory rhythm. Further, it was show that an agent-model that embeds this hypothesis reproduces a surprising number of taxis signatures observed in larvae. Also, by coupling the sensory input to a neural oscillator in continuous time, it was shown that the mechanism is robust and biologically plausible. The mechanism provides a simple architecture for combining information across modalities, and explaining how learnt associations modulate taxis. The results in the light of larval neural circuitry are discussed, and testable predictions are made.
Ichinose, T. and Tanimoto, H. (2016). Dynamics of memory-guided choice behavior in Drosophila. Proc Jpn Acad Ser B Phys Biol Sci 92: 346-357. PubMed ID: 27725473
Memory retrieval requires both accuracy and speed. Olfactory learning of the fruit fly Drosophila melanogaster serves as a powerful model system to identify molecular and neuronal substrates of memory and memory-guided behavior. The behavioral expression of olfactory memory has traditionally been tested as a conditioned odor response in a simple T-maze, which measures the result, but not the speed, of odor choice. This study developed multiplexed T-mazes that allow video recording of the choice behavior. Automatic fly counting in each arm of the maze visualizes choice dynamics. Using this setup, it was shown that the transient blockade of serotonergic neurons slows down the choice, while leaving the eventual choice intact. In contrast, activation of the same neurons impairs the eventual performance leaving the choice speed unchanged. This new apparatus contributes to elucidating how the speed and the accuracy of memory retrieval are implemented in the fly brain.

Friday, November 25th

Hayashi, R., Schnabl, J., Handler, D., Mohn, F., Ameres, S. L. and Brennecke, J. (2016). Genetic and mechanistic diversity of piRNA 3'-end formation. Nature [Epub ahead of print]. PubMed ID: 27851737
Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI-interacting RNAs (piRNAs)-the 22-30-nt-long guides for PIWI-clade Ago proteins that silence transposons in animal gonads-are generated independently of Dicer from single-stranded precursors. piRNA 5' ends are defined either by Zucchini, the Drosophila homologue of mitoPLD - a mitochondria-anchored endonuclease, or by piRNA - guided target cleavage. Formation of piRNA 3' ends is poorly understood. This study report that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub) or Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler (Drosophila homologue of Mut-7). The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. These data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3'-end formation.
Ghezzi, A., Zomeno, M., Pietrzykowski, A. Z. and Atkinson, N. S. (2016). Immediate-early alcohol-responsive miRNA expression in Drosophila. J Neurogenet: 1-10. PubMed ID: 27845601
At the core of the changes characteristic of alcoholism are alterations in gene expression in the brain of the addicted individual. These changes are believed to underlie some of the neuroadaptations that promote compulsive drinking. Unfortunately, the mechanisms by which alcohol consumption produces changes in gene expression remain poorly understood. MicroRNAs (miRNAs) have emerged as important regulators of gene expression because they can coordinately modulate the translation efficiency of large sets of specific mRNAs. This study investigated the early miRNA responses elicited by an acute sedating dose of alcohol in the Drosophila model organism. In this analysis, the power of next-generation sequencing was combined with Drosophila genetics to identify alcohol-sensitive miRNAs and to functionally test them for a role in modulating alcohol sensitivity. Fourteen known Drosophila miRNAs, and 13 putative novel miRNAs were identified that respond to an acute sedative exposure to alcohol. Using the GeneSwitch Gal4/UAS system, a subset of these ethanol-responsive miRNAs was functionally tested to determine their individual contribution in modulating ethanol sensitivity. Two microRNAs were identified that when overexpressed significantly increased ethanol sensitivity: miR-6 and miR-310. MicroRNA target prediction analysis revealed that the different alcohol-responsive miRNAs target-overlapping sets of mRNAs. Alcoholism is the product of accumulated cellular changes produced by chronic ethanol consumption. Although all of the changes described here are extremely rapid responses evoked by a single ethanol exposure, understanding the gene expression changes that occur in the first few minutes after ethanol exposure will help categorization of ethanol responses into those that are near instantaneous and those that are emergent responses produced only by repeated ethanol exposure.
Cerro-Herreros, E., Fernandez-Costa, J.M., Sabater-Arcis, M., Llamusi, B. and Artero, R. (2016). Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila. Sci Rep 6: 36230. PubMed ID: 27805016
Myotonic Dystrophy type 1 (DM1) originates from alleles of the DMPK gene with hundreds of extra CTG repeats in the 3' untranslated region (3' UTR). CUG repeat RNAs accumulate in foci that sequester Muscleblind-like (MBNL) proteins away from their functional target transcripts. Endogenous upregulation of MBNL proteins is, thus, a potential therapeutic approach to DM1. This study identifies two miRNAs, dme-miR-277 and dme-miR-304, that differentially regulate muscleblind RNA isoforms in miRNA sensor constructs. It was shown that their sequestration by sponge constructs derepresses endogenous muscleblind not only in a wild type background but also in a DM1 Drosophila model expressing non-coding CUG trinucleotide repeats throughout the musculature. Enhanced muscleblind expression results in significant rescue of pathological phenotypes, including reversal of several mis-splicing events and reduced muscle atrophy in DM1 adult flies. Rescued flies have improved muscle function in climbing and flight assays, and have longer lifespan compared to disease controls. These studies provide proof of concept for a similar potentially therapeutic approach to DM1 in humans.

Black, K, L., Petruk, S., Fenstermaker, T. K., Hodgson, J. W., Caplan, J. L., Brock, H. W. and Mazo, A. (2016). Chromatin proteins and RNA are associated with DNA during all phases of mitosis. Cell Discov 2: 16038. PubMed ID: 27807477
Mitosis brings about major changes to chromosome and nuclear structure. This study used recently developed proximity ligation assay-based techniques to investigate the association with DNA of chromatin-associated proteins and RNAs in Drosophila embryos during mitosis. All groups of tested proteins, histone-modifying and chromatin-remodeling proteins and methylated histones remained in close proximity to DNA during all phases of mitosis. RNA transcripts were found to be associated with DNA during all stages of mitosis. Reduction of H3K27me3 levels or elimination of RNAs had no effect on the association of the components of PcG and TrxG complexes to DNA. Using a combination of proximity ligation assay-based techniques and super-resolution microscopy, he number of protein-DNA and RNA-DNA foci was found to undergo significant reduction during mitosis, suggesting that mitosis may be accompanied by structural re-arrangement or compaction of specific chromatin domains.

Funikov, S. Y., Ryazansky, S. S., Kanapin, A. A., Logacheva, M. D., Penin, A. A., Snezhkina, A. V., Shilova, V. Y., Garbuz, D. G., Evgen'ev, M. B. and Zatsepina, O. G. (2016). Interplay between RNA interference and heat shock response systems in Drosophila melanogaster. Open Biol 6. PubMed ID: 27805906
The genome expression pattern is strongly modified during the heat shock response (HSR) to form an adaptive state. This may be partly achieved by modulating microRNA levels that control the expression of a great number of genes that are embedded within the gene circuitry. This study investigated the cross-talk between two highly conserved and universal house-keeping systems, the HSR and microRNA machinery, in Drosophila melanogaster. Pronounced interstrain differences in the microRNA levels are alleviated after heat shock (HS) to form a uniform microRNA pattern. However, individual strains exhibit different patterns of microRNA expression during the course of recovery. Importantly, HS-regulated microRNAs may target functionally similar HS-responsive genes involved in the HSR. Despite the observed general downregulation of primary microRNA precursor expression as well as core microRNA pathway genes after HS, the levels of many mature microRNAs are upregulated. This indicates that the regulation of miRNA expression after HS occurs at transcriptional and post-transcriptional levels. Deletion of all hsp70 genes had no significant effect on microRNA biogenesis but might influence the dynamics of microRNA expression during the HSR.
Yang CY, Ramamoorthy S, Boller S, Rosenbaum M, Rodriguez Gil A, Mittler G, Imai Y, Kuba K, Grosschedl R. (2016). Interaction of CCR4-NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis. Genes Dev. [Epub ahead of print]. PubMed ID: 27807034
Evolutionary Homolog Study
Transcription factor EBF1 (early B-cell factor 1; see Drosophila Collier/Knot) regulates early B-cell differentiation by poising or activating lineage-specific genes and repressing genes associated with alternative cell fates. SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry of was used to identify proteins associated with endogenous EBF1 in pro-B cells. This analysis identified most components of the multifunctional CCR4-NOT complex, which regulates transcription and mRNA degradation. CNOT3 interacts with EBF1, and histidine 240 in EBF1 was identified as a critical residue for this interaction. Complementation of Ebf1-/- progenitors with EBF1H240A revealed a partial block of pro-B-cell differentiation and altered expression of specific EBF1 target genes that show either reduced transcription or increased mRNA stability. Most deregulated EBF1 target genes show normal occupancy by EBF1H240A, but genes were also detected with altered occupancy, suggesting that the CCR4-NOT complex affects multiple activities of EBF1. Mice with conditional Cnot3 inactivation recapitulate the block of early B-cell differentiation, which was found to be associated with an impaired autoregulation of Ebf1 and reduced expression of pre-B-cell receptor components. Thus, the interaction of the CCR4-NOT complex with EBF1 diversifies the function of EBF1 in a context-dependent manner and may coordinate transcriptional and post-transcriptional gene regulation.

Thursday, November 24th

Yue, Y., Ke, S., Zhou, W. and Chang, J. (2016). In vivo imaging reveals composite coding for diagonal motion in the Drosophila visual system. PLoS One 11: e0164020. PubMed ID: 27695103
Understanding information coding is important for resolving the functions of visual neural circuits. The motion vision system is a classic model for studying information coding as it contains a concise and complete information-processing circuit. In Drosophila, the axon terminals of motion-detection neurons (T4 and T5) project to the lobula plate, which comprises four regions that respond to the four cardinal directions of motion. The lobula plate thus represents a topographic map on a transverse plane. This enables study of the coding of diagonal motion by investigating its response pattern. By using in vivo two-photon calcium imaging, the axon terminals of T4 and T5 cells in the lobula plate were found to be activated during diagonal motion. Further experiments showed that the response to diagonal motion is distributed over the following two regions compared to the cardinal directions of motion-a diagonal motion selective response region and a non-selective response region-which overlap with the response regions of the two vector-correlated cardinal directions of motion. Interestingly, the sizes of the non-selective response regions are linearly correlated with the angle of the diagonal motion. These results revealed that the Drosophila visual system employs a composite coding for diagonal motion that includes both independent coding and vector decomposition coding.
Buhl, E., Bradlaugh, A., Ogueta, M., Chen, K. F., Stanewsky, R. and Hodge, J. J. (2016). Quasimodo mediates daily and acute light effects on Drosophila clock neuron excitability. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27821737
The light-input factor Quasimodo (Qsm) regulates rhythmic electrical excitability of clock neurons, presumably via an Na+, K+, Cl- cotransporter (NKCC) and the Shaw K+ channel (dKV3.1). Because of light-dependent degradation of the clock protein Timeless (Tim), constant illumination (LL) leads to a breakdown of molecular and behavioral rhythms. Both overexpression (OX) and knockdown (RNAi) of qsm, NKCC, or Shaw led to robust LL rhythmicity. Whole-cell recordings of the large ventral lateral neurons (l-LNv) showed that altering Qsm levels reduced the daily variation in neuronal activity: qsmOX led to a constitutive less active, night-like state, and qsmRNAi led to a more active, day-like state. Qsm also affected daily changes in K+ currents and the GABA reversal potential, suggesting a role in modifying membrane currents and GABA responses in a daily fashion, potentially modulating light arousal and input to the clock. When directly challenged with blue light, wild-type l-LNvs responded with increased firing at night and no net response during the day, whereas altering Qsm, NKKC, or Shaw levels abolished these day/night differences. Finally, coexpression of ShawOX and NKCCRNAi in a qsm mutant background restored LL-induced behavioral arrhythmicity and wild-type neuronal activity patterns, suggesting that the three genes operate in the same pathway. It is proposed that Qsm affects both daily and acute light effects in l-LNvs probably acting on Shaw and NKCC.
Li, J., Zhang, W., Guo, Z., Wu, S., Jan, L.Y. and Jan, Y.N. (2016). A defensive kicking behavior in response to mechanical stimuli mediated by Drosophila wing margin bristles. J Neurosci 36: 11275-11282. PubMed ID: 27807168
Mechanosensation, one of the fastest sensory modalities, mediates diverse behaviors including those pertinent for survival. It is important to understand how mechanical stimuli trigger defensive behaviors. This study reports that Drosophila melanogaster adult flies exhibit a kicking response against invading parasitic mites over their wing margin with ultrafast speed and high spatial precision. Mechanical stimuli that mimic the mites' movement evoke a similar kicking behavior. Further, a TRPV channel, Nanchung, and a specific Nanchung-expressing neuron under each recurved bristle that forms an array along the wing margin were identified as being essential sensory components for this behavior. Electrophysiological recordings demonstrate that the mechanosensitivity of recurved bristles requires Nanchung and Nanchung-expressing neurons. Together, these results reveal a novel neural mechanism for innate defensive behavior through mechanosensation.

Turner, H.N., Armengol, K., Patel, A.A., Himmel, N.J., Sullivan, L., Iyer, S.C., Bhattacharya, S., Iyer, E.P., Landry, C., Galko, M.J. and Cox, D.N. (2016). The TRP channels Pkd2, NompC, and Trpm act in cold-sensing neurons to mediate unique aversive behaviors to noxious cold in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 27818173
The basic mechanisms underlying noxious cold perception are not well understood. This study developed Drosophila assays for noxious cold responses. Larvae respond to near-freezing temperatures via a mutually exclusive set of singular behaviors-in particular, a full-body contraction (CT). Class III (CIII) multidendritic sensory neurons are specifically activated by cold and optogenetic activation of these neurons elicits CT. Blocking synaptic transmission in CIII neurons inhibits CT. Genetically, the transient receptor potential (TRP) channels Trpm, NompC, and Polycystic kidney disease 2 (Pkd2) are expressed in CIII neurons, where each is required for CT. Misexpression of Pkd2 is sufficient to confer cold responsiveness. The optogenetic activation level of multimodal CIII neurons determines behavioral output, and visualization of neuronal activity supports this conclusion. Coactivation of cold- and heat-responsive sensory neurons suggests that the cold-evoked response circuitry is dominant. This Drosophila model will enable a sophisticated molecular genetic dissection of cold nociceptive genes and circuits.

Wednesday, November 23rd

Wu, C. H., Zong, Q., Du, A. L., Zhang, W., Yao, H. C., Yu, X. Q. and Wang, Y. F. (2016). Knockdown of Dynamitin in testes significantly decreased male fertility in Drosophila melanogaster. Dev Biol [Epub ahead of print]. PubMed ID: 27742209
Dynamitin (Dmn) is a major component of dynactin, a multiprotein complex playing important roles in a variety of intracellular motile events. Wolbachia bacterial infection has been shown to result in a reduction of Dmn protein. As Wolbachia may modify sperm in male hosts, it is speculated that Dmn may have a function in male fertility. Knockdown of Dmn in testes dramatically decreased male fertility, overexpression of Dmn in Wolbachia-infected males significantly rescued male fertility, indicating an important role of Dmn in inducing male fertility defects following Wolbachia infection. Some scattered immature sperm with late canoe-shaped head distributed in the end of Dmn knockdown testis and only about half mature sperm were observed in the Dmn knockdown testis relative to those in the control. Immunofluorescence staining showed significantly less abundance of tubulin around the nucleus of spermatid and scattered F-actin cones to different extents in the individualization complex (IC) during spermiogenesis in Dmn knockdown testes, which may disrupt the nuclear condensation and sperm individualization. Since dynein-dynactin complex has been shown to mediate transport of many cellular components, including mRNAs and organelles, these results suggest that Dmn may play an important role in Drosophila spermiogenesis by affecting transport of many important cytoplasmic materials.
Lian, G., Dettenhofer, M., Lu, J., Downing, M., Chenn, A., Wong, T. and Sheen, V. (2016). FilaminA and Formin2 dependent endocytosis regulates proliferation via the canonical Wnt pathway. Development [Epub ahead of print]. PubMed ID: 27789627
Evolutionary Homolog Study
Actin-associated proteins regulate multiple cellular processes, including proliferation and differentiation, but the molecular mechanisms underlying these processes are unclear. This study reports that the actin-binding protein FilaminA (see Drosophila Cheerio) physically interacts with the actin-nucleating protein Formin2 (see Drosophila Daam). Loss of FilaminA and Formin2 impairs proliferation, thereby generating multiple embryonic phenotypes, including microcephaly. FilaminA interacts with the Wnt co-receptor Lrp6. Loss of FilaminA and Formin2 impairs Lrp6 endocytosis, downstream Gsk3β (see Drosophila Shaggy) activity, and β-catenin (see Drosophila Armadillo) accumulation in the nucleus. Finally, the proliferative defect in null FilaminA and Formin2 neural progenitors is rescued by inhibiting Gsk3β activity. These findings provide an unrealized mechanism whereby actin-associated endocytosis regulates proliferation by mediating molecules in the canonical Wnt pathway. Moreover, the Formin2-dependent signaling in this pathway parallels that seen in the non-canonical Wnt-dependent regulation of planar cell polarity through the Formin homology Daam protein. These studies provide evidence for integration of actin-associated processes in directing neuroepithelial proliferation.
Duque, J. and Gorfinkiel, N. (2016). Integration of actomyosin contractility with cell-cell adhesion during dorsal closure. Development [Epub ahead of print]. PubMed ID: 27836966
This work combined genetic perturbation, time-lapse imaging and quantitative image analysis to investigate how pulsatile actomyosin contractility drives cell oscillations, apical cell contraction and tissue closure, during the morphogenesis of the amnioserosa, the main force-generating tissue during the process of Dorsal Closure in Drosophila. This work reveals that Myosin activity determines the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. In contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, in amnioserosa cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through the control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis.
Jurado, J., de Navascues, J. and Gorfinkiel, N. (2016). α-Catenin stabilises Cadherin-Catenin complexes and modulates actomyosin dynamics to allow pulsatile apical contraction. J Cell Sci [Epub ahead of print]. PubMed ID: 27831494
This study investigated how cell contractility and adhesion are functionally integrated during epithelial morphogenesis. To this end,the role of α-Catenin, a key molecule linking E-Cadherin-based adhesion and the actomyosin cytoskeleton, during Drosophila embryonic dorsal closure, by studying a newly developed allelic series. α-Catenin was shown to regulate pulsatile apical contraction in the amnioserosa, the main force-generating tissue driving closure of the embryonic epidermis. α-Catenin controls actomyosin dynamics by stabilising and promoting the formation of actomyosin foci, and also stabilises DE-Cadherin at the cell membrane, suggesting that medioapical actomyosin contractility regulates junction stability. Furthermore, this study uncovered a genetic interaction between α-Catenin and Vinculin, and a tension-dependent recruitment of Vinculin to amniosersoa apical cell membranes, suggesting the existence of a mechano-sensitive module operating in this tissue.

Bremer J and Granato M. (2016). Myosin phosphatase fine-tunes zebrafish motoneuron position during axonogenesis. PLoS Genet. 12(11):e1006440. PubMed ID: 27855159
Evolutionary Homolog Study
During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, this study shows that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. This alignment requires myosin phosphatase activity (see Drosophila Pp1-87B) within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin (see Drosophila Zipper) phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, this study demonstrates that spinal motoneurons fine-tune their position during axonogenesis and the myosin II regulatory network was identified as a key regulator.
Zhu Z, et al. (2016). Functional Coordination of WAVE and WASP in C. elegans Neuroblast Migration. Dev Cell 39(2):224-238. PubMed ID: 27780040
Evolutionary Homolog Study
Directional cell migration is critical for metazoan development. This study defines two molecular pathways that activate the Arp2/3 complex during neuroblast migration in Caenorhabditis elegans. The transmembrane protein MIG-13/Lrp12 is linked to the Arp2/3 nucleation-promoting factors WAVE (see Drosophila SCAR) or WASP (see Drosophila WASp) through direct interactions with ABL-1 (see Drosophila Abl) or SEM-5/Grb2 (see Drosophila Drk), respectively. WAVE mutations partially impaired F-actin organization and decelerated cell migration, and WASP mutations did not inhibit cell migration but enhanced migration defects in WAVE-deficient cells. Purified SEM-5 and MIG-2 synergistically stimulated the F-actin branching activity of WASP-Arp2/3 in vitro. In GFP knockin animals, WAVE and WASP were largely organized into separate clusters at the leading edge, and the amount of WASP was less than WAVE but could be elevated by WAVE mutations. These results indicate that the MIG-13-WAVE pathway provides the major force for directional cell motility, whereas MIG-13-WASP partially compensates for its loss, underscoring their coordinated activities in facilitating robust cell migration.

Tuesday, November 22nd

Wei, Y., Gokhale, R. H., Sonnenschein, A., Montgomery, K. M., Ingersoll, A. and Arnosti, D. N. (2016). Complex cis-regulatory landscape of the insulin receptor gene underlies the broad expression of a central signaling regulator. Development 143: 3591-3603. PubMed ID: 27702787
Insulin signaling plays key roles in development, growth and metabolism through dynamic control of glucose uptake, global protein translation and transcriptional regulation. Altered levels of insulin signaling are known to play key roles in development and disease, yet the molecular basis of such differential signaling remains obscure. Expression of the insulin receptor (InR) gene itself appears to play an important role, but the nature of the molecular wiring controlling InR transcription has not been elucidated. This study characterized the regulatory elements driving Drosophila InR expression and found that the generally broad expression of this gene is belied by complex individual switch elements, the dynamic regulation of which reflects direct and indirect contributions of FOXO, EcR, Rbf and additional transcription factors through redundant elements dispersed throughout approximately 40 kb of non-coding regions. The control of InR transcription in response to nutritional and tissue-specific inputs represents an integration of multiple cis-regulatory elements, the structure and function of which may have been sculpted by evolutionary selection to provide a highly tailored set of signaling responses on developmental and tissue-specific levels.
Zhou, Q., Yu, L., Friedrich, M. and Pignoni, F. (2016). Distinct regulation of atonal in a visual organ of Drosophila: organ-specific enhancer and lack of autoregulation in the larval eye. Dev Biol [Epub ahead of print]. PubMed ID: 27693434
Drosophila has three types of visual organs, the larval eyes or Bolwig's organs (BO), the ocelli (OC) and the compound eyes (CE). In all, the bHLH protein Atonal (Ato) functions as the proneural factor for photoreceptors and effects the transition from progenitor cells to differentiating neurons. This work investigates the regulation of ato expression in the BO primordium (BOP). Surprisingly, atotranscription in the BOP was found to be is entirely independent of the shared regulatory DNA for the developing CE and OC. The core enhancer for BOP expression, atoBO, lies ~6kb upstream of the ato gene, in contrast to the downstream location of CE and OC regulatory elements. Moreover, maintenance of ato expression in the neuronal precursors through autoregulation-a common and ancient feature of ato expression that is well-documented in eyes, ocelli and chordotonal organs-does not occur in the BO. The atoBO enhancer contains two binding sites for the transcription factor Sine oculis (So), a core component of the progenitor specification network in all three visual organs. These binding sites function in vivo and are specifically bound by So in vitro. Taken together, these findings reveal that the control of ato transcription in the evolutionarily derived BO has diverged considerably from ato regulation in the more ancestral compound eyes and ocelli, to the extent of acquiring what appears to be a distinct and evolutionarily novel cis-regulatory module.
Francis, M. J., Roche, S., Cho, M. J., Beall, E., Min, B., Panganiban, R. P. and Rio, D. C. (2016). Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27799520
In Drosophila, P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein-DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that is termed "Inverted Repeat Binding Protein (IRBP) 18" and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability.
Zhang Y, Zhang L, Tang X, Bhardwaj SR, Ji J, Rong YS (2016). MTV, an ssDNA protecting complex essential for transposon-based telomere maintenance in Drosophila. PLoS Genet. 2016 Nov 11;12(11):e1006435. PubMed ID: 27835648
Multiple complexes protect telomeres. In telomerase-maintained organisms, Shelterin related complexes occupy the duplex region while the CST and Tpp1-Pot1 complexes bind the single stranded overhang of telomeres. Drosophila uses a transposon-based mechanism for end protection. The HOAP-HipHop complex occupies the duplex region. Whether an ssDNA-binding complex exists is not known. This study discovered a novel protein, Tea, that is specifically enriched at telomeres to prevent telomere fusion. A complex was identified consisting of Tea and two known capping proteins, Ver and Moi. The Moi-Tea-Ver (MTV) complex purified in vitro binds and protects ssDNA in a sequence-independent manner. Tea recruits Ver and Moi to telomeres, and point mutations disrupting MTV interaction in vitro result in telomere uncapping, consistent with these proteins functioning as a complex in vivo. MTV thus shares functional similarities with CST or TPP1-POT1 in protecting ssDNA, highlighting a conserved feature in end protecting mechanisms.

Sun C, Jiang L, Liu Y, Shen H, Weiss SJ, Zhou Y, Rui L. (2016). Adipose Snail1 regulates lipolysis and lipid partitioning by suppressing Adipose Triacylglycerol Lipase expression. Cell Rep 17(8):2015-2027. PubMed ID: 27851965
Evolutionary Homolog Study
Lipolysis provides metabolic fuel; however, aberrant adipose lipolysis results in ectopic lipid accumulation and lipotoxicity. While adipose triacylglycerol lipase (ATGL) (see Drosophila Brummer) catalyzes the first step of lipolysis, its regulation is not fully understood. This study demonstrated that adipocyte Snail1 (see Drosophila Snail) suppresses both ATGL expression and lipolysis. Adipose Snail1 levels are higher in fed mice than in fasted mice and higher in obese mice as opposed to lean mice. Insulin increases Snail1 levels in both murine and human adipocytes, wherein Snail1 binds to the ATGL promoter to repress its expression. Importantly, adipocyte-specific deletion of Snail1 increases adipose ATGL expression and lipolysis, resulting in decreased fat mass and increased liver fat content in mice fed either a normal chow diet or a high-fat diet. Thus, this study has identified a Snail1-ATGL axis that regulates adipose lipolysis and fatty acid release, thereby governing lipid partitioning between adipose and non-adipose tissues.
Mandoli A, et al. (2016). The Hematopoietic transcription factors RUNX1 and ERG prevent AML1-ETO oncogene overexpression and onset of the apoptosis program in t(8;21) AMLs. Cell Rep. 17(8):2087-2100. PubMed ID: 27851970
Evolutionary Homolog Study
The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. This study investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels of histone acetylation but also at distal elements characterized by low acetylation levels and binding of the hematopoietic bHLH transcription factor LYL1 and LMO2 (see Drosophila Beadex). In contrast, ETS transcription factor ERG, ETS transcription factor FLI1, TAL1, and RUNX1 (see Drosophila Runt) bind at all AML1-ETO-occupied regulatory regions, including those of the AML1-ETO gene itself, suggesting their involvement in regulating AML1-ETO expression levels. While expression of AML1-ETO in myeloid differentiated induced pluripotent stem cells (iPSCs) induces leukemic characteristics, overexpression increases cell death. Expression of wild-type transcription factors RUNX1 and ERG in AML was found to be required to prevent this oncogene overexpression. Together these results show that the interplay of the epigenome and transcription factors prevents apoptosis in t(8;21) AML cells.

Monday, November 21st

Field, A., Xiang, J., Anderson, W. R., Graham, P. and Pick, L. (2016). Activation of Ftz-F1-responsive genes through Ftz/Ftz-F1 dependent enhancers. PLoS One 11: e0163128. PubMed ID: 27723822
The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern.
Papadopoulos, D. K., Krmpot, A. J., Nikolic, S. N., Krautz, R., Terenius, L., Tomancak, P., Rigler, R., Gehring, W. J. and Vukojevic, V. (2015). Probing the kinetic landscape of Hox transcription factor-DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy. Mech Dev 138 Pt 2: 218-225. PubMed ID: 26428533
Hox genes encode transcription factors that control the formation of body structures, segment-specifically along the anterior-posterior axis of metazoans. Hox transcription factors bind nuclear DNA pervasively and regulate a plethora of target genes, deploying various molecular mechanisms that depend on the developmental and cellular context. To analyze quantitatively the dynamics of their DNA-binding behavior, confocal laser scanning microscopy (CLSM), single-point fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS) and bimolecular fluorescence complementation (BiFC) were used. The Hox transcription factor Sex combs reduced (Scr) was shown to form dimers that strongly associate with its specific fork head binding site (fkh250) in live salivary gland cell nuclei. In contrast, dimers of a constitutively inactive, phospho-mimicking variant of Scr show weak, non-specific DNA-binding. These studies reveal that nuclear dynamics of Scr is complex, exhibiting a changing landscape of interactions that is difficult to characterize by probing one point at a time. Therefore, mechanistic evidence is provided using massively parallel FCS (mpFCS). Scr dimers were found to predominantly formed on the DNA and are equally abundant at the chromosomes and an introduced multimeric fkh250 binding-site, indicating different mobilities, presumably reflecting transient binding with different affinities on the DNA. These proof-of-principle results emphasize the advantages of mpFCS for quantitative characterization of fast dynamic processes in live cells.
Li, J., Chauve, L., Phelps, G., Brielmann, R. M. and Morimoto, R. I. (2016). E2F coregulates an essential HSF developmental program that is distinct from the heat-shock response. Genes Dev 30: 2062-2075. PubMed ID: 27688402
Evolutionary Homolog Study
Heat-shock factor (HSF; see Drosophila Hsf) is the master transcriptional regulator of the heat-shock response (HSR) and is essential for stress resilience. HSF is also required for metazoan development; however, its function and regulation in this process are poorly understood. This study characterize the genomic distribution and transcriptional activity of Caenorhabditis elegans HSF-1 during larval development and showed that the developmental HSF-1 transcriptional program is distinct from the HSR. HSF-1 developmental activation requires binding of E2F/DP (see Drosophila E2f) to a GC-rich motif that facilitates HSF-1 binding to a heat-shock element (HSE) that is degenerate from the consensus HSE sequence and adjacent to the E2F-binding site at promoters. In contrast, induction of the HSR is independent of these promoter elements or E2F/DP and instead requires a distinct set of tandem canonical HSEs. Together, E2F and HSF-1 directly regulate a gene network, including a specific subset of chaperones, to promote protein biogenesis and anabolic metabolism, which are essential in development.
Gueta, K., et al. (2016). The stage-dependent roles of Ldb1 and functional redundancy with Ldb2 in mammalian retinogenesis. Development [Epub ahead of print]. PubMed ID: 27697904
Evolutionary Homolog Study
The Lim domain binding proteins (Ldbs; see Drosophila Chip) are key cofactor proteins that assemble with LIM domains of the LMO/LIM-HD family to form functional complexes that regulate cell proliferation and differentiation throughout the CNS. Ldb1 interacts with Lhx2 (see Drosophila Apterous) in the embryonic mouse retina, and both Ldb1 and Ldb2, probably functioning with Lhx2 in a complex, play a key role in maintaining the pool of retinal progenitor cells. This is accomplished by controlling the expression of the homeodomain factors Vsx2 (see Drosophila Vsx1) and Rax, as well as components of the Notch and Hedgehog signaling pathways. Furthermore, the Ldb1/Ldb2 mediated complex, is essential for generation of early-born photoreceptors through the regulation of Rax and Crx (an Orthodenticle homolog). Finally, functional redundancy between Ldb1 and Ldb2 was demonstrated. Ldb1 can fully compensate the loss of Ldb2 during all phases of retinal development, whereas Ldb2 alone is sufficient to sustain activity of Lhx2 in both early and late-stage RPCs and in Muller glia. In contrast, loss of Ldb1 disrupts activity of the LIM domain factors in neuronal precursors. These findings uncover an intricate regulatory network mediated by Ldb1 and Ldb2 that promotes RPC proliferation and multipotency, and also controls specification of the major cell types of the mammalian retina.

Sunday, November 20th

Wang, A., Mouser, J., Pitt, J., Promislow, D. and Kaeberlein, M. (2016). Rapamycin enhances survival in a Drosophila model of mitochondrial disease. Oncotarget [Epub ahead of print]. PubMed ID: 27741510
Pediatric mitochondrial disorders are a devastating category of diseases caused by deficiencies in mitochondrial function. Leigh Syndrome (LS) is the most common of these diseases with symptoms typically appearing within the first year of birth and progressing rapidly until death, usually by 6-7 years of age. Genetic inhibition of the mechanistic target of rapamycin (TOR; see Drosophila TOR) has been shown to rescue the short lifespan of yeast mutants with defective mitochondrial function, and pharmacological inhibition of TOR by administration of rapamycin significantly rescues the shortened lifespan, neurological symptoms, and neurodegeneration in a mouse model of LS. However, the mechanism by which TOR inhibition exerts these effects, and the extent to which these effects can extend to other models of mitochondrial deficiency, are unknown. This study probed the effects of TOR inhibition in a Drosophila model of complex I deficiency. Treatment with rapamycin robustly suppresses the lifespan defect in this model of LS, without affecting behavioral phenotypes. Interestingly, this increased lifespan in response to TOR inhibition occurs in an autophagy-independent manner. Further, a fat storage defect was detected in mitochondrial-DNA-encoded NADH dehydrogenase subunit 2 (ND2) mutant flies that is rescued by rapamycin, supporting a model that rapamycin exerts its effects on mitochondrial disease in these animals by altering metabolism.
Baril, C., Gavory, G., Bidla, G., Knaevelsrud, H., Sauvageau, G. and Therrien, M. (2016). Human NUP98-HOXA9 promotes hyperplastic growth of hematopoietic tissues in Drosophila. Dev Biol [Epub ahead of print]. PubMed ID: 27838340
Acute myeloid leukemia (AML) is a complex malignancy with poor prognosis. Several genetic lesions can lead to the disease. One of these corresponds to the NUP98-HOXA9 (NA9) translocation that fuses sequences encoding the N-terminal part of NUP98 (see Drosophila Nucleoporin 98-96kD) to those encoding the DNA-binding domain of HOXA9. Despite several studies, the mechanism underlying NA9 ability to induce leukemia is still unclear. To bridge this gap, this study sought to functionally dissect NA9 activity using Drosophila. For this, transgenic NA9 fly lines were generated and the oncoprotein was expressed during larval hematopoiesis. This markedly enhanced cell proliferation and tissue growth, but did not alter cell fate specification. Moreover, reminiscent to NA9 activity in mammals, strong cooperation was observed between NA9 and the MEIS homolog HTH. Genetic characterization of NA9-induced phenotypes suggested interference with PVR (Flt1-4 RTK homolog) signaling, which is similar to functional interactions observed in mammals between Flt3 and HOXA9 in leukemia. Finally, NA9 expression was also found to induce non-cell autonomous effects, raising the possibility that its leukemia-inducing activity also relies on this property. Together, this work suggests that NA9 ability to induce blood cell expansion is evolutionarily conserved. The amenability of NA9 activity to a genetically-tractable system should facilitate unraveling its molecular underpinnings.
Hasan, M. N., Hosen, M. J., Thakur, P. K., Abir, R. A., Zubaer, A., Renkai, G., Yoshida, M., Ohta, H., Lee, J. M., Kusakabe, T. and Hirashima, A. (2016). In vitro screening for inhibitor of cloned Drosophila melanogaster tyramine-β-hydroxylase and docking studies. Int J Biol Macromol 93: 889-895. PubMed ID: 27355756
Biogenic amines are common biologically active substances extended within the whole animal kingdom where they play vital roles as signal transducer as well as regulator of cell functions. One of these biogenic amines called octopamine (OA) is synthesized from tyramine (TA) by the catalysis of tyramine-β-hydroxylase (TβH) originated in the insect nervous system. Both TA and OA act as neurotransmitters, neurohormones and neuromodulators in the arthropod nervous system. In this study, the inhibitory activity of 1-arylimidazole-2(3H)-thiones (AITs) was tested on cloned Drosophila tyramine-β-hydroxylase (DmTβH) expressed in Bombyx mori strain. Radiolabelled 3H-TA was used to analyze the activity of AITs exhibited inhibitory effects on DmTβH, whose ID50 values range from 0.02 to 2511nM where DmTβH was inhibited in a dose-dependent manner at pH 7.6 and 25 ° C during a 30min of incubation. To understand the catalytic role of the TβH, a three dimensional structure of the TβH from Drosophila melanogaster was constructed by homology modeling using the Phyre2 web server with 100% confidence. The modeled three-dimensional structure of TβH was used to perform the docking study with AITs. This may give more insights to precise design of inhibitors for TβH to control insect's population.
Cruz-Becerra, G., Juarez, M., Valadez-Graham, V. and Zurita, M. (2016). Analysis of Drosophila p8 and p52 mutants reveals distinct roles for the maintenance of TFIIH stability and male germ cell differentiation. Open Biol 6 [Epub ahead of print] PubMed ID: 27805905
Eukaryotic gene expression is activated by factors that interact within complex machinery to initiate transcription. An important component of this machinery is the DNA repair/transcription factor TFIIH. Mutations in TFIIH result in three human syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Transcription and DNA repair defects have been linked to some clinical features of these syndromes. However, how mutations in TFIIH affect specific developmental programmes, allowing organisms to develop with particular phenotypes, is not well understood. This study shows that mutations in the p52 and p8 subunits of TFIIH have a moderate effect on the gene expression programme in the Drosophila testis, causing germ cell differentiation arrest in meiosis, but no Polycomb enrichment at the promoter of the affected differentiation genes, supporting recent data that disagree with the current Polycomb-mediated repression model for regulating gene expression in the testis. Moreover, TFIIH stability was not compromised in p8 subunit-depleted testes that show transcriptional defects, highlighting the role of p8 in transcription. Therefore, this study reveals how defects in TFIIH affect a specific cell differentiation programme and contributes to understanding the specific syndrome manifestations in TFIIH-afflicted patients.

Kandul, N. P., Zhang, T., Hay, B. A. and Guo, M. (2016). Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila. Nat Commun 7: 13100. PubMed ID: 27841259
Mitochondrial DNA (mtDNA) often exists in a state of heteroplasmy, in which mutant mtDNA co-exists in cells with wild-type mtDNA. High frequencies of pathogenic mtDNA result in maternally inherited diseases; maternally and somatically acquired mutations also accumulate over time and contribute to diseases of ageing. Reducing heteroplasmy is therefore a therapeutic goal and in vivo models in post-mitotic tissues are needed to facilitate these studies. This study describes a transgene-based model of a heteroplasmic lethal mtDNA deletion (mtDNAΔ) in adult Drosophila muscle. Stimulation of autophagy, activation of the PINK1/parkin pathway or decreased levels of mitofusin result in a selective decrease in mtDNAΔ. Decreased levels of mitofusin and increased levels of ATPIF1, an inhibitor of ATP synthase reversal-dependent mitochondrial repolarization, result in a further decrease in mtDNAΔ levels. These results show that an adult post-mitotic tissue can be cleansed of a deleterious genome, suggesting that therapeutic removal of mutant mtDNA can be achieved.
Altanbyek, V., Cha, S. J., Kang, G. U., Im, D. S., Lee, S., Kim, H. J. and Kim, K. (2016). Imbalance of mitochondrial dynamics in Drosophila models of amyotrophic lateral sclerosis. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 27810362
Amyotrophic lateral sclerosis (ALS) is the most common neurodegenerative disease, characterized by progressive and selective loss of motor neurons in the brain and spinal cord. DNA/RNA-binding proteins such as TDP-43 (see Drosophila TAR DNA-binding protein-43 homolog), FUS (see Drosophila Cabeza), and TAF15 have been linked with the sporadic and familial forms of ALS. However, the exact pathogenic mechanism of ALS is still unknown. Recent studies have found that ALS-causing genes such as TDP-43, FUS, and TAF15 genetically interact with mitochondrial dynamics regulatory genes. This study shows that mitochondrial fission was highly enhanced in muscles and motor neurons of TDP-43, FUS, and TAF15-induced fly models of ALS. Furthermore, the mitochondrial fission defects were rescued by co-expression of mitochondrial dynamics regulatory genes such as Marf, Opa1, and the dominant negative mutant form of Drp1. Moreover, the expression level of Marf was decreased in ALS-induced flies. These results indicate that the imbalance of mitochondrial dynamics caused by instability of Marf is linked to the pathogenesis of TDP-43, FUS, and TAF15-associated ALS.

Saturday, November 19th

Li, Y., Hoffmann, J., Li, Y., Stephano, F., Bruchhaus, I., Fink, C. and Roeder, T. (2016). Octopamine controls starvation resistance, life span and metabolic traits in Drosophila. Sci Rep 6: 35359. PubMed ID: 27759117
The monoamines octopamine (OA) and tyramine (TA) modulate numerous behaviours and physiological processes in invertebrates. Nevertheless, it is not clear whether these invertebrate counterparts of norepinephrine are important regulators of metabolic and life history traits. This study shows that flies (Drosophila melanogaster) lacking OA are more resistant to starvation, while their overall life span is substantially reduced compared with control flies. In addition, these animals have increased body fat deposits, reduced physical activity and a reduced metabolic resting rate. Increasing the release of OA from internal stores induced the opposite effects. Flies devoid of both OA and TA had normal body fat and metabolic rates, suggesting that OA and TA act antagonistically. Moreover, OA-deficient flies show increased insulin release rates. It was inferred that the OA-mediated control of insulin release accounts for a substantial proportion of the alterations observed in these flies. Apparently, OA levels control the balance between thrifty and expenditure metabolic modes. Thus, changes in OA levels in response to external and internal signals orchestrate behaviour and metabolic processes to meet physiological needs. Moreover, chronic deregulation of the corresponding signalling systems in humans may be associated with metabolic disorders, such as obesity or diabetes.
Yerushalmi, G. Y., Misyura, L., Donini, A. and MacMillan, H. A. (2016). Chronic dietary salt stress mitigates hyperkalemia and facilitates chill coma recovery in Drosophila melanogaster. J Insect Physiol 95: 89-97. PubMed ID: 27642001
Chill susceptible insects like Drosophila lose the ability to regulate water and ion homeostasis at low temperatures. This loss of hemolymph ion and water balance drives a hyperkalemic state that depolarizes cells, causing cellular injury and death. The ability to maintain ion homeostasis at low temperatures and/or recover ion homeostasis upon rewarming is closely related to insect cold tolerance. It was hypothesized that changes to organismal ion balance, which can be achieved in Drosophila through dietary salt loading, could alter whole animal cold tolerance phenotypes. Flies were put in the presence of diets highly enriched in NaCl, KCl, xylitol (an osmotic control) or sucrose (a dietary supplement known to impact cold tolerance) for 24h. Independently of their osmotic effects, NaCl, KCl, and sucrose supplementation all improved the ability of flies to maintain K+ balance in the cold, which allowed for faster recovery from chill coma after 6h at 0 ° C. These supplements, however, also slightly increased the CTmin and had little impact on survival rates following chronic cold stress (24h at 0 ° C), suggesting that the effect of diet on cold tolerance depends on the measure of cold tolerance assessed. In contrast to prolonged salt stress, brief feeding (1.5h) on diets high in salt slowed coma recovery, suggesting that the long-term effects of NaCl and KCl on chilling tolerance result from phenotypic plasticity, induced in response to a salty diet, rather than simply the presence of the diet in the gut lumen.
Luo, J., Shen, W. L. and Montell, C. (2016). TRPA1 mediates sensation of the rate of temperature change in Drosophila larvae. Nat Neurosci [Epub ahead of print]. PubMed ID: 27749829
Avoidance of noxious ambient heat is crucial for survival. A well-known phenomenon is that animals are sensitive to the rate of temperature change. However, the cellular and molecular underpinnings through which animals sense and respond much more vigorously to fast temperature changes are unknown. Using Drosophila larvae, this study found that nociceptive rolling behavior was triggered at lower temperatures and at higher frequencies when the temperature increased rapidly. Neurons in the brain were identified that were sensitive to the speed of the temperature increase rather than just to the absolute temperature. These cellular and behavioral responses depended on the TRPA1 channel, whose activity responded to the rate of temperature increase. It is proposed that larvae use low-threshold sensors in the brain to monitor rapid temperature increases as a protective alert signal to trigger rolling behaviors, allowing fast escape before the temperature of the brain rises to dangerous levels.
Maniere, G., Ziegler, A. B., Geillon, F., Featherstone, D. E. and Grosjean, Y. (2016). Direct sensing of nutrients via a LAT1-like transporter in Drosophila insulin-producing cells. Cell Rep 17: 137-148. PubMed ID: 27681427
Dietary leucine has been suspected to play an important role in insulin release, a hormone that controls satiety and metabolism. The mechanism by which insulin-producing cells (IPCs) sense leucine and regulate insulin secretion is still poorly understood. In Drosophila, insulin-like peptides (DILP2 and DILP5) are produced by brain IPCs and are released in the hemolymph after leucine ingestion. Using Ca(2+)-imaging and ex vivo cultured larval brains, IPCs were shown to directly sense extracellular leucine levels via minidiscs (MND), a leucine transporter. MND knockdown in IPCs abolished leucine-dependent changes, including loss of DILP2 and DILP5 in IPC bodies, consistent with the idea that MND is necessary for leucine-dependent DILP release. This, in turn, leads to a strong increase in hemolymph sugar levels and reduced growth. GDH knockdown in IPCs also reduced leucine-dependent DILP release, suggesting that nutrient sensing is coupled to the glutamate dehydrogenase pathway.
Peng, G., Kashio, M., Li, T., Dong, X., Tominaga, M. and Kadowaki, T. (2016). TRPA1 channels in Drosophila and honey bee ectoparasitic mites share heat sensitivity and temperature-related physiological functions. Front Physiol 7: 447. PubMed ID: 27761115
Evolutionary Homolog Study
TRPA1 is conserved between many arthropods, and in some has been shown to function as a chemosensor for noxious compounds. Activation of arthropod TRPA1 channels by temperature fluctuations has been tested in only a few insect species, and all of them were shown to be activated by heat. The recent identification of chemosensitive TRPA1 channels from two honey bee ectoparasitic mite species (VdTRPA1 and TmTRPA1) have provided an opportunity to study the temperature-dependent activation and the temperature-associated physiological functions of TRPA1 channels in non-insect arthropods. This study found that both mite TRPA1 channels are heat sensitive and capable of rescuing the temperature-related behavioral defects of a Drosophila melanogaster trpA1 mutant. These results suggest that heat-sensitivity of TRPA1 could be conserved between many arthropods despite its amino acid sequence diversity. Nevertheless, the ankyrin repeats (ARs) 6 and 7 are well-conserved between six heat-sensitive arthropod TRPA1 channels and have critical roles for the heat activation of VdTRPA1.
Asaoka, T., Almagro, J., Ehrhardt, C., Tsai, I., Schleiffer, A., Deszcz, L., Junttila, S., Ringrose, L., Mechtler, K., Kavirayani, A., Gyenesei, A., Hofmann, K., Duchek, P., Rittinger, K. and Ikeda, F. (2016). Linear ubiquitination by LUBEL has a role in Drosophila heat stress response. EMBO Rep 17: 1624-1640. PubMed ID: 27702987
The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. This study provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila. This study identified Drosophila CG11321, which was renamed Linear Ubiquitin E3 ligase (LUBEL), and found that it catalyzes linear ubiquitination in vitro. Endogenous linear ubiquitin chain-derived peptides were detected by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, linear ubiquitination-defective flies were established by mutating residues essential for the catalytic activity of LUBEL. Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.

Friday, November 19th

Wu, S., Joseph, A., Hammonds, A. S., Celniker, S. E., Yu, B. and Frise, E. (2016). Stability-driven nonnegative matrix factorization to interpret spatial gene expression and build local gene networks. Proc Natl Acad Sci U S A 113: 4290-4295. PubMed ID: 27071099
Spatial gene expression patterns enable the detection of local covariability and are extremely useful for identifying local gene interactions during normal development. The abundance of spatial expression data in recent years has led to the modeling and analysis of regulatory networks. The inherent complexity of such data makes it a challenge to extract biological information. This paper describes staNMF, a method that combines a scalable implementation of nonnegative matrix factorization (NMF) with a new stability-driven model selection criterion. When applied to a set of Drosophila early embryonic spatial gene expression images, one of the largest datasets of its kind, staNMF identified 21 principal patterns (PP). Providing a compact yet biologically interpretable representation of Drosophila expression patterns, PP are comparable to a fate map generated experimentally by laser ablation and show exceptional promise as a data-driven alternative to manual annotations. This analysis mapped genes to cell-fate programs and assigned putative biological roles to uncharacterized genes. Finally, the PP was used to generate local transcription factor regulatory networks. Spatially local correlation networks were constructed for six PP, giant, hunchback, knirps, Kruppel, huckebein, and tailless, that span along the embryonic anterior-posterior axis. Using a two-tail 5% cutoff on correlation, 10 of the 11 links were reproduced in the well-studied gap gene network. The performance of PP with the Drosophila data suggests that staNMF provides informative decompositions and constitutes a useful computational lens through which to extract biological insight from complex and often noisy gene expression data.
Yasuoka, Y., Shinzato, C. and Satoh, N. (2016). The mesoderm-forming gene brachyury regulates ectoderm-endoderm demarcation in the coral Acropora digitifera. Curr Biol [Epub ahead of print]. PubMed ID: 27693135
Evolutionary Homolog Study
Blastoporal expression of the T-box transcription factor gene brachyury (see Drosophila Brachyenteron) is conserved in most metazoans. Cnidarians are basal metazoans that are important for understanding evolution of metazoan body plans. Because they lack mesoderm, they have been used to investigate the evolutionary origins of mesoderm. This study focused on corals, a primitive clade of cnidarians that diverged from sea anemones approximately 500 mya. A microinjection method for coral eggs to was developed to examine Brachyury functions during embryogenesis of the scleractinian coral, Acropora digitifera. Because Acropora embryos undergo pharynx formation after the blastopore closes completely, they are useful to understand Brachyury functions in gastrulation movement and pharynx formation. Blastoporal expression of brachyury is directly activated by Wnt/β-catenin signaling (see Drosophila Wingless pathway) in the ectoderm of coral embryos, indicating that the regulatory axis from Wnt/β-catenin signaling to brachyury is highly conserved among eumetazoans. Loss-of-function analysis demonstrated that Brachyury is required for pharynx formation but not for gastrulation movement. Genome-wide transcriptome analysis demonstrated that genes positively regulated by Brachyury are expressed in the ectoderm of Acropora gastrulae, while negatively regulated genes are in endoderm. Therefore, germ layer demarcation around the blastopore appears to be the evolutionarily conserved role of Brachyury during gastrulation. Compared with Brachyury functions in vertebrate mesoderm-ectoderm and mesoderm-endoderm demarcation, the results suggest that the vertebrate-type mesoderm may have originated from brachyury-expressing ectoderm adjacent to endoderm.
Mbodj, A., Gustafson, E. H., Ciglar, L., Junion, G., Gonzalez, A., Girardot, C., Perrin, L., Furlong, E. E. and Thieffry, D. (2016). Qualitative dynamical modelling can formally explain mesoderm specification and predict novel developmental phenotypes. PLoS Comput Biol 12: e1005073. PubMed ID: 27599298
Given the complexity of developmental networks, it is often difficult to predict the effect of genetic perturbations, even within coding genes. Regulatory factors generally have pleiotropic effects, exhibit partially redundant roles, and regulate highly interconnected pathways with ample cross-talk. This study delineated a logical model encompassing 48 components and 82 regulatory interactions involved in mesoderm specification during Drosophila development, thereby providing a formal integration of all available genetic information from the literature. The four main tissues derived from mesoderm, heart, somatic muscles, visceral mesoderm and fat body correspond to alternative stable states. The model can predict known mutant phenotypes and use it to systematically predict the effects of over 300 new, often non-intuitive, loss- and gain-of-function mutations, and combinations thereof. Several novel predictions were further validated experimentally, thereby demonstrating the robustness of model. Logical modelling can thus contribute to formally explain and predict regulatory outcomes underlying cell fate decisions.
Miao, G. and Hayashi, S. (2016). Escargot controls the sequential specification of two tracheal tip cell types by suppressing FGF signaling in Drosophila. Development [Epub ahead of print]. PubMed ID: 27742749
Extrinsic branching factors promote the elongation and migration of tubular organs. In the Drosophila tracheal system, Branchless/Fibroblast Growth Factor (FGF) stimulates the branching program by specifying tip cells that acquire motility and lead branch migration to a specific destination. Tip cells have two alternative cell fates: the terminal cell (TC), which elongates the cytoplasmic extension with intracellular lumen, and the fusion cell (FC), which mediates branch connections to form tubular networks. How Branchless/FGF controls this specification of cells with distinct shapes and behaviors is unknown. This study reports that this cell-type diversification involves the modulation of FGF signaling by the zinc-finger protein Escargot (Esg), which is expressed in the FC and is essential for its specification. The dorsal branch begins elongation with a pair of tip cells with high FGF signaling. When the branch tip reaches its final destination, one of the tip cells become an FC and expresses Esg. FCs and TCs differ in their response to FGF: TCs are attracted by FGF, while FCs are repelled. Esg suppresses ERK signaling in FCs to control this differential migratory behavior.

Urbansky, S., Gonzalez Avalos, P., Wosch, M. and Lemke, S. (2016). Folded gastrulation and T48 drive the evolution of coordinated mesoderm internalization in flies. Elife 5 [Epub ahead of print]. PubMed ID: 27685537
Evolutionary Homolog Study
Gastrulation constitutes a fundamental yet diverse morphogenetic process of metazoan development. Modes of gastrulation range from stochastic translocation of individual cells to coordinated infolding of an epithelial sheet. This study identified two genes, folded gastrulation and t48, which in the evolution of Drosophila gastrulation acted as a likely switch from an ingression of individual cells to the invagination of the blastoderm epithelium. Both genes are expressed and required for mesoderm invagination in Drosophila but do not appear during mesoderm ingression of the midge Chironomus riparius. Early expression of either or both of these genes in C.riparius is sufficient to invoke mesoderm invagination similar to D.melanogaster. The possible genetic simplicity and a measurable increase in developmental robustness might explain repeated evolution of similar transitions in animal gastrulation.
Lu, H., Sokolow, A., Kiehart, D. P. and Edwards, G. S. (2016). Quantifying dorsal closure in three dimensions. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27798232
Dorsal closure is an essential stage of Drosophila embryogenesis and is a powerful model system for morphogenesis, wound healing, and tissue biomechanics. During closure two flanks of lateral epidermis close an eye-shaped dorsal opening that is filled with amnioserosa. The two flanks of lateral epidermis are zipped together at each canthus ("corner" of the eye). Actomyosin-rich purse strings are localized at each of the two leading edges of lateral epidermis ("lids" of the eye). This study reports that each purse string indents the dorsal surface at each leading edge. The amnioserosa tissue bulges outward during the early-to-mid stages of closure to form a remarkably smooth, asymmetric dome indicative of an isotropic and uniform surface tension. Internal pressure of the embryo and tissue elastic properties help shape the dorsal surface.

Thursday, November 17th

Montague, S. A. and Baker, B. S. (2016). Memory elicited by courtship conditioning requires mushroom body neuronal subsets similar to those utilized in appetitive memory. LoS One 11: e0164516. PubMed ID: 27764141
In Drosophila courtship conditioning, male flies learn not to court females during training with an unreceptive female. He retains a memory of this training and for several hours decreases courtship when subsequently paired with any female. Courtship conditioning is a unique learning paradigm; it uses a positive-valence stimulus, a female fly, to teach a male to decrease an innate behavior, courtship of the female. As such, courtship conditioning is not clearly categorized as either appetitive or aversive conditioning. The mushroom body (MB) region in the fruit fly brain is important for several types of memory; however, the precise subsets of intrinsic and extrinsic MB neurons necessary for courtship conditioning are unknown. This study disrupted synaptic signaling by driving a shibirets effector in precise subsets of MB neurons, defined by a collection of split-GAL4 drivers. Out of 75 lines tested, 32 showed defects in courtship conditioning memory. Surprisingly, there were no hits in the γ lobe Kenyon cells, a region previously implicated in courtship conditioning memory. Several γ lobe extrinsic neurons were necessary for courtship conditioning memory. Overall, the memory hits in the dopaminergic neurons (DANs) and the mushroom body output neurons were more consistent with results from appetitive memory assays than aversive memory assays. For example, protocerebral anterior medial DANs were necessary for courtship memory, similar to appetitive memory, while protocerebral posterior lateral 1 (PPL1) DANs, important for aversive memory, were not needed. Overall, these results indicate that the MB circuits necessary for courtship conditioning memory coincide with circuits necessary for appetitive memory.
Jiang, Y., Pitmon, E., Berry, J., Wolf, F. W., McKenzie, Z. and Lebestky, T. J. (2016). A genetic screen to assess Dopamine receptor (DopR1) dependent sleep regulation in Drosophila. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27760793
Sleep is an essential behavioral state of rest that is regulated by homeostatic drives to ensure a balance of sleep and activity, as well as independent arousal mechanisms in the central brain. Dopamine has been identified as a critical regulator of both sleep behavior and arousal. This study presents results of a genetic screen that selectively restored the Dopamine Receptor (DopR/DopR1/dumb) to specific neuroanatomical regions of the adult Drosophila brain to assess requirements for DopR in sleep behavior. Subsets of the mushroom body were identified that utilize DopR in daytime sleep regulation. These data are supported by multiple examples of spatially restricted genetic rescue data in discrete circuits of the mushroom body, as well as immunohistochemistry that corroborates the localization of DopR protein within mushroom body circuits. Independent loss of function data using an inducible RNAi construct in the same specific circuits also supports a requirement for DopR in daytime sleep. Additional circuit activation of discrete DopR+ mushroom body neurons also suggests roles for these subpopulations in sleep behavior. These conclusions support a new separable function for DopR in daytime sleep regulation within the mushroom body. This daytime regulation is independent of the known role of DopR in nighttime sleep, which is regulated within the Fan Shaped Body. This study provides new neuroanatomical loci for exploration of dopaminergic sleep functions in Drosophila, and expands understanding of sleep regulation during the day versus night.
Pankova, K. and Borst, A. (2016). RNA-seq transcriptome analysis of direction-selective T4/T5 neurons in Drosophila. PLoS One 11: e0163986. PubMed ID: 27684367
Neuronal computation underlying detection of visual motion has been studied for more than a half-century. In Drosophila, direction-selective T4/T5 neurons show supralinear signal amplification in response to stimuli moving in their preferred direction, in agreement with the prediction made by the Hassenstein-Reichardt detector. Nevertheless, the molecular mechanism explaining how the Hassenstein-Reichardt model is implemented in T4/T5 cells has not been identified yet. The present study utilized cell type-specific transcriptome profiling with RNA-seq to obtain a complete gene expression profile of T4/T5 neurons. The expression was analyzed of genes that affect neuronal computational properties and can underlie the molecular implementation of the core features of the Hassenstein-Reichardt model to the dendrites of T4/T5 neurons. Furthermore, the acquired RNA-seq data was used to examine the neurotransmitter system used by T4/T5 neurons. Surprisingly, co-expression of the cholinergic markers and the vesicular GABA transporter was observed in T4/T5 neurons. Previously undetected expression of vesicular GABA transporter was documented in T4/T5 cells using VGAT-LexA knock-in line. The provided gene expression dataset can serve as a useful source for studying the properties of direction-selective T4/T5 neurons on the molecular level.
Salazar-Gatzimas, E., Chen, J., Creamer, M. S., Mano, O., Mandel, H. B., Matulis, C. A., Pottackal, J. and Clark, D. A. (2016). Direct measurement of correlation responses in Drosophila elementary motion detectors reveals fast timescale tuning. Neuron 92: 227-239. PubMed ID: 27710784
Animals estimate visual motion by integrating light intensity information over time and space. The integration requires nonlinear processing, which makes motion estimation circuitry sensitive to specific spatiotemporal correlations that signify visual motion. Classical models of motion estimation weight these correlations to produce direction-selective signals. However, the correlational algorithms they describe have not been directly measured in elementary motion-detecting neurons (EMDs). This study employed stimuli to directly measure responses to pairwise correlations in Drosophila's EMD neurons, T4 and T5. Activity in these neurons was required for behavioral responses to pairwise correlations and was predictive of those responses. The pattern of neural responses in the EMDs was inconsistent with one classical model of motion detection, and the timescale and selectivity of correlation responses constrained the temporal filtering properties in potential models. These results reveal how neural responses to pairwise correlations drive visual behavior in this canonical motion-detecting circuit.
Stratmann, J., Gabilondo, H., Benito-Sipos, J. and Thor, S. (2016). Neuronal cell fate diversification controlled by sub-temporal action of Kruppel. Elife 5 [Epub ahead of print]. PubMed ID: 27740908
During Drosophila embryonic nervous system development, neuroblasts express a programmed cascade of five temporal transcription factors that govern the identity of cells generated at different time-points. However, these five temporal genes fall short of accounting for the many distinct cell types generated in large lineages. This study finds that the late temporal gene castor sub-divides its large window in neuroblast 5-6 by simultaneously activating two cell fate determination cascades and a sub-temporal regulatory program. The sub-temporal program acts both upon itself and upon the determination cascades to diversify the castor window. Surprisingly, the early temporal gene Kruppel acts as one of the sub-temporal genes within the late castor window. Intriguingly, while the temporal gene castor activates the two determination cascades and the sub-temporal program, spatial cues controlling cell fate in the latter part of the 5-6 lineage exclusively act upon the determination cascades.
Kundu, S., Ganguly, A., Chakraborty, T. S., Kumar, A. and Siddiqi, O. (2016). Synergism and combinatorial coding for binary odor mixture perception in Drosophila. eNeuro 3 [Epub ahead of print]. PubMed ID: 27588303
Most odors in the natural environment are mixtures of several compounds. Olfactory receptors housed in the olfactory sensory neurons detect these odors and transmit the information to the brain, leading to decision-making. But whether the olfactory system detects the ingredients of a mixture separately or treats mixtures as different entities is not well understood. Using Drosophila melanogaster as a model system, this study has demonstrated that fruit flies perceive binary odor mixtures in a manner that is heavily dependent on both the proportion and the degree of dilution of the components, suggesting a combinatorial coding at the peripheral level. This coding strategy appears to be receptor specific and is independent of interneuronal interactions.

Wednesday, November 16th

Li, M., Lindblad, J. L., Perez, E., Bergmann, A. and Fan, Y. (2016). Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation. BMC Biol 14: 70. PubMed ID: 27542914
ATG1 belongs to the Uncoordinated-51-like kinase protein family. Members of this family are best characterized for roles in macroautophagy and neuronal development. Apoptosis-induced proliferation (AiP) is a caspase-directed and JNK-dependent process which is involved in tissue repair and regeneration after massive stress-induced apoptotic cell loss. Under certain conditions, AiP can cause tissue overgrowth with implications for cancer. This study shows that Atg1 in Drosophila (dAtg1) has a previously unrecognized function for both regenerative and overgrowth-promoting AiP in eye and wing imaginal discs. dAtg1 acts genetically downstream of and is transcriptionally induced by JNK activity, and it is required for JNK-dependent production of mitogens such as Wingless for AiP. Interestingly, this function of dAtg1 in AiP is independent of its roles in autophagy and in neuronal development. It is concluded that, in addition to a role of dAtg1 in autophagy and neuronal development, a third function of dAtg1 for AiP is reported.
Subasic, D., Stoeger, T., Eisenring, S., Matia-Gonzalez, A. M., Imig, J., Zheng, X., Xiong, L., Gisler, P., Eberhard, R., Holtackers, R., Gerber, A. P., Pelkmans, L. and Hengartner, M. O. (2016). Post-transcriptional control of executioner caspases by RNA-binding proteins. Genes Dev 30: 2213-2225. PubMed ID: 27798844
Evolutionary Homolog Study
Regulation of caspases occurs at several levels, including transcription, proteolytic processing, inhibition of enzymatic function, and protein degradation. In contrast, little is known about the extent of post-transcriptional control of caspases. This study describes four conserved RNA-binding proteins (RBPs)-PUF-8, MEX-3, GLD-1, and CGH-1-that sequentially repress the CED-3 caspase (see Drosophila DREDD) in distinct regions of the Caenorhabditis elegans germline. GLD-1 represses ced-3 mRNA translation via two binding sites in its 3' untranslated region (UTR), thereby ensuring a dual control of unwanted cell death: at the level of p53/CEP-1 and at the executioner caspase level. Moreover, seven RBPs were identified that regulate human caspase-3 expression and/or activation, including human PUF-8, GLD-1, and CGH-1 homologs PUM1, QKI, and DDX6. Given the presence of unusually long executioner caspase 3' UTRs in many metazoans, translational control of executioner caspases by RBPs might be a strategy used widely across the animal kingdom to control apoptosis.
Liu, D., Shaukat, Z., Xu, T., Denton, D., Saint, R. and Gregory, S. (2016). Autophagy regulates the survival of cells with chromosomal instability. Oncotarget [Epub ahead of print]. PubMed ID: 27590505
Chromosomal instability (CIN) refers to genomic instability in which cells have gained or lost chromosomes or chromosomal fragments. A high level of CIN is common in solid tumours and is associated with cancer drug resistance and poor prognosis. The impact of CIN-induced stress and the resulting cellular responses are only just beginning to emerge. Using proliferating tissue in Drosophila as a model, this study found that autophagy is activated in CIN cells and is necessary for their survival. Specifically, increasing the removal of defective mitochondria by mitophagy is able to lower levels of reactive oxygen species and the resultant cellular damage that is normally seen in CIN cells. In response to DNA damage, CIN is increased in a positive feedback loop, and increasing autophagy by Tor depletion was found to decrease the level of CIN in proliferating cells. These findings underline the importance of autophagy control in the development of CIN tumours.
Braden, C. R. and Neufeld, T. P. (2016). Atg1-independent induction of autophagy by the Drosophila Ulk3 homolog ADUK. FEBS J [Epub ahead of print]. PubMed ID: 27717182
Although canonical autophagy regulation requires a multi-protein complex centered on the Ser/Thr-kinase Atg1 (mammalian Ulk1/2), alternative signals can induce autophagy independent of Atg1 through unknown mechanisms. This study identified the Drosophila Ulk3 ortholog, another Drosophila Unc-51-like kinase (ADUK: CG8866), as an Atg1-independent autophagy inducer. ADUK interacts with Atg1 complex members Atg13 and 200 kDa FAK family kinase-interacting protein (FIP200 or Atg17), and requires Atg13 but not Atg1 for autophagy induction. Loss of ADUK shortens adult lifespan and reduces the autophagic response to a chemical stressor, dimethyl sulfoxide. However, ADUK is not required for autophagy induction by Atg1-dependent nutrient or developmental cues. Atg1 and ADUK/Ulk3 thus represent alternative catalytic components of a shared autophagy kinase complex.

Tuesday, November 15th

Bahk, S. and Jones, W. D. (2016). Insect odorant receptor trafficking requires calmodulin. BMC Biol 14: 83. PubMed ID: 27686128
Like most animals, insects rely on their olfactory systems for finding food and mates and in avoiding noxious chemicals and predators. Most insect olfactory neurons express an odorant-specific odorant receptor (OR) along with Orco, the olfactory co-receptor. Orco binds ORs and permits their trafficking to the dendrites of antennal olfactory sensory neurons (OSNs), where together, they are suggested to form heteromeric ligand-gated non-selective cation channels. While most amino acid residues in Orco are well conserved across insect orders, one especially well-conserved region in Orco's second intracellular loop is a putative calmodulin (CaM) binding site (CBS). This study, exploreed the relationship between Orco and CaM in vivo in the olfactory neurons of Drosophila melanogaster. OSN-specific knock-down of CaM at the onset of OSN development was found to disrupt the spontaneous firing of OSNs and reduce Orco trafficking to the ciliated dendrites of OSNs without affecting their morphology. A series of Orco CBS mutant proteins was generated and found that none of them rescue the Orco-null Orco 1 mutant phenotype, which is characterized by an OR protein trafficking defect that blocks spontaneous and odorant-evoked OSN activity. In contrast to an identically constructed wild-type form of Orco that does rescue the Orco 1 phenotype, all the Orco CBS mutants remain stuck in the OSN soma, preventing even the smallest odorant-evoked response. Last, it was found that CaM's modulation of OR trafficking is dependent on activity. Knock-down of CaM in all Orco-positive OSNs after OR expression is well established has little effect on olfactory responsiveness alone. When combined with an extended exposure to odorant, however, this late-onset CaM knock-down significantly reduces both olfactory sensitivity and the trafficking of Orco only to the ciliated dendrites of OSNs that respond to the exposed odorant. In conclusion that study has show CaM regulates OR trafficking and olfactory responses in vivo in Drosophila olfactory neurons via a well-conserved binding site on the olfactory co-receptor Orco. As CaM's modulation of Orco seems to be dependent on activity, a model is proposed in which the CaM/Orco interaction allows insect OSNs to maintain appropriate dendritic levels of OR regardless of environmental odorant concentrations.
Paulsen, R., Bahner, M. and Huber, A. (2000). The PDZ assembled "transducisome" of microvillar photoreceptors: the TRP/TRPL problem. Pflugers Arch 439: R181-R183. PubMed ID: 27757613
Two types of ion channels, TRP and TRPL, are activated upon light-absorption in rhabdomeral photoreceptor membranes of fly compound eyes. Whereas TRP is associated with other signaling proteins into a multiprotein complex (transducisome), the molecular organization of TRPL is discussed controversely. This study analysed the TRPL content of blowfly rhabdomeral membranes and investigated by co-immunoprecipitation studies whether or not TRPL is part of the transducisome. Compared to TRP there are at least ten times less TRPL molecules present in the rhabdomeral membrane. A small fraction of the total TRPL present co-immunoprecipitates with other proteins of the transducisome and vice versa. These data suggest that a significant fraction of TRPL is not incorporated into the transducisome. This fraction may either form independent ion channels or bind to the transducisome transiently.

Asaoka, T., et al. (2016). Linear ubiquitination by LUBEL has a role in Drosophila heat stress response. EMBO Rep [Epub ahead of print]. PubMed ID: 27702987
The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. This study provides the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila. This study identified Drosophila CG11321, which was named Linear Ubiquitin E3 ligase (LUBEL), and it was found to catalyze linear ubiquitination in vitro. Endogenous linear ubiquitin chain-derived peptides were detected by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, linear ubiquitination-defective flies were established by mutating residues essential for the catalytic activity of LUBEL Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.
Li, H. B., Wang, R. X., Jiang, H. B., Zhang, E. D., Tan, J. Q., Xu, H. Z., Zhou, R. R. and Xia, X. B. (2016). Mitochondrial ribosomal Protein L10 Associates with Cyclin B1/Cdk1 Activity and Mitochondrial Function. DNA Cell Biol. PubMed ID: 27726420
Mitochondria ribosomal proteins are important for mitochondrial-encoded protein synthesis and mitochondrial function. In addition to their roles in mitoribosome assembly, several mitochondrial ribosome proteins are also implicated in cellular processes like cell cycle regulation, apoptosis, and mitochondrial homeostasis regulation. This study demonstrate that MRPL10 regulates cyclin B1/Cdk1 (cyclin-dependent kinase 1) activity and mitochondrial protein synthesis in mammalian cells. In Drosophila, inactivation of mRpL10 (the Drosophila ortholog of mammalian MRPL10) in eyes results in abnormal eye development. Furthermore, expression of human cyclin B1 suppresses eye phenotypes and mitochondrial abnormality of mRpL10 knockdown Drosophila. This study identified that the physiological regulatory pathway of MRPL10 and providing new insights into the role of MRPL10 in growth control and mitochondrial function.
Strilbytska, O. M., Semaniuk, U. V., Storey, K. B., Edgar, B. A. and Lushchak, O. V. (2016). Activation of the Tor/Myc signaling axis in intestinal stem and progenitor cells affects longevity, stress resistance and metabolism in Drosophila.. Comp Biochem Physiol B Biochem Mol Biol 203: 92-99. PubMed ID: 27693629
The TOR (target of rapamycin) signaling pathway and the transcriptional factor Myc play important roles in growBh control. Myc acts, in part, as a downstream target of TOR to regulate the activity and functioning of stem cells. Tbis study explored the role of TOR-Myc axis in stem and progenitor cells in the regulation of lifespan, stress resistance and metabolism in Drosophila. Goth overexpression of rheb and myc-rheb in midgut stem and progenitor cells decreased the lifespan and starvation resistance of flies. TOR activation caused higher survival under malnutrition conditions. Furthermore, gut-specific activation of JAK/STAT and insulin signaling pathways were demonstrated to control gut integrity. Both genetic manipulations had an impact on carbohydrate metabolism and transcriptional levels of metabolic genes. These findings indicate that activation of the TOR-Myc axis in midgut stem and progenitor cells influences a variety of traits in Drosophila.

Monday, October 14th

Schiesari, L., Andreatta, G., Kyriacou, C. P., O'Connor, M. B. and Costa, R. (2016). The Insulin-Like proteins dILPs-2/5 determine diapause inducibility in Drosophila. PLoS One 11: e0163680. PubMed ID: 27689881
Diapause is an actively induced dormancy that has evolved in Metazoa to resist environmental stresses. In temperate regions, many diapausing insects overwinter at low temperatures by blocking embryonic, larval or adult development. Despite its Afro-tropical origin, Drosophila melanogaster migrated to temperate regions of Asia and Europe where females overwinter as adults by arresting gonadal development (reproductive diapause) at temperatures <13 degrees C. Recent work in D. melanogaster has implicated the developmental hormones dILP-2 and/or dILP3, and dILP5, homologues of vertebrate insulin/insulin-like growth factors (IGFs), in reproductive arrest. However, polymorphisms in timeless (tim) and couch potato (cpo) dramatically affect diapause inducibility and these dILP experiments could not exclude this common genetic variation contributing to the diapause phenotype. This study applied an extensive genetic dissection of the insulin signaling pathway which facilitates seeing both enhancements and reductions in egg development that are independent of tim and cpo variations. A number of manipulations dramatically enhance diapause to ~100%. These include ablating, or reducing the excitability of the insulin-producing cells (IPCs) that express dILPs-2,3,5 employing the dilp2,3,5-/- triple mutant, desensitizing insulin signaling using a chico mutation, or inhibiting dILP2 and 5 in the hemolymph by over-expressing Imaginal Morphogenesis Protein-Late 2 (Imp-L2). In addition, triple mutant dilp2,3,5-/- females maintain high levels of diapause even when temperatures are raised in adulthood to 19 ° C. However at 22 ° C, these females all show egg development revealing that the effects are conditional on temperature and not a general female sterility. In contrast, over-expression of dilps-2/5 or enhancing IPC excitability, led to levels of ovarian arrest that approached zero, underscoring dILPs-2 and 5 as key antagonists of diapause.
de Miguel, C., Linsler, F., Casanova, J. and Franch-Marro, X. (2016). Genetic basis for the evolution of organ morphogenesis. The case of spalt and cut in development of insect trachea. Development [Epub ahead of print]. PubMed ID: 27578790
Changes in body organ morphology have allowed animals to better exploit diverse habitats. As morphogenesis in general and organogenesis in particular are under genetic control, genetic modifications provide the basis for a wide range of morphologies. Knowledge of the genetic basis of phenotypic diversification in evolution has focused mostly on quantitative traits. However, it is not clear how simple genetic changes can account for the coordinated variations that give rise to modified functional organs. This study addressed this issue by analysing the expression and function of regulatory genes in the developing tracheal systems of two insect species. The larval tracheal system of Drosophila can be distinguished from the less derived tracheal system of the beetle Tribolium by two main features. First, the lateral spiracles, which in Tribolium connect the tracheal branches to the exterior in each segment, are not present in Drosophila. Instead, Drosophila has only one pair of strongly derived posterior spiracles. Second, the dorsal trunks, two prominent branches that distribute air from the posterior spiracles and extend longitudinally through the larva, are not present in Tribolium. Both innovations, while considered different structures, are functionally dependent on each other and linked to habitat occupancy. In this regard, buried Drosophila larvae in semi-liquid environments keep their posterior spiracles above the surface and distribute the gas along the body via the dorsal trunks. Conversely, the lateral spiracles of free-living Tribolium larvae provide sufficient airflow to all segments making unnecessary the formation of thick dorsal trunks. This study shows that changes in the domains of spalt and cut expression are associated with the acquisition of each innovation. Moreover, these two genetic modifications are connected both functionally and genetically, thus providing an evolutionary scenario by which a genetic event contributes to the joint evolution of functionally interrelated structures.
Nam, S. C. (2016). Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis. Genesis [Epub ahead of print]. PubMed ID: 27579500
Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Tau, a microtubule associated protein, was analyzed in this study to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par-1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. Tau was found to have a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation.
Aghajanian, P., Takashima, S., Paul, M., Younossi-Hartenstein, A. and Hartenstein, V. (2016). Metamorphosis of the Drosophila visceral musculature and its role in intestinal morphogenesis and stem cell formation. Dev Biol [Epub ahead of print]. PubMed ID: 27765651
This study has combined the use of specific markers with electron microscopy to follow the formation of the adult visceral musculature and its involvement in gut development during metamorphosis. Unlike the adult somatic musculature, which is derived from a pool of undifferentiated myoblasts, the visceral musculature of the adult is a direct descendant of the larval fibers, as shown by activating a lineage tracing construct in the larval muscle and obtaining labeled visceral fibers in the adult. However, visceral muscles undergo a phase of remodeling that coincides with the metamorphosis of the intestinal epithelium. During the first day following puparium formation, both circular and longitudinal syncytial fibers dedifferentiate, losing their myofibrils and extracellular matrix, and dissociating into mononuclear cells ("secondary myoblasts"). Towards the end of the second day, this process is reversed, and between 48 and 72h after puparium formation, a structurally fully differentiated adult muscle layer has formed. The musculature, the intestinal epithelium is completely renewed during metamorphosis. The adult midgut epithelium rapidly expands over the larval layer during the first few hours after puparium formation; in case of the hindgut, replacement takes longer, and proceeds by the gradual caudad extension of a proliferating growth zone, the hindgut proliferation zone (HPZ). The subsequent elongation of the hindgut and midgut, as well as the establishment of a population of intestinal stem cells active in the adult midgut and hindgut, requires the presence of the visceral muscle layer, based on the finding that ablation of this layer causes a severe disruption of both processes.

Sunday, November 13th

Podratz, J. L., Lee, H., Knorr, P., Koehler, S., Forsythe, S., Lambrecht, K., Arias, S., Schmidt, K., Steinhoff, G., Yudintsev, G., Yang, A., Trushina, E. and Windebank, A. (2016). Cisplatin induces mitochondrial deficits in Drosophila larval segmental nerve. Neurobiol Dis [Epub ahead of print]. PubMed ID: 27765583
Cisplatin is an effective chemotherapy drug that induces peripheral neuropathy in cancer patients. In rodent dorsal root ganglion neurons, cisplatin binds nuclear and mitochondrial DNA (mtDNA) inducing DNA damage and apoptosis. Platinum-mtDNA adducts inhibit mtDNA replication and transcription leading to mitochondrial degradation. Cisplatin also induces climbing deficiencies associated with neuronal apoptosis in adult Drosophila. This study used Drosophila larvae that express GFP in the mitochondria of motor neurons to observe the effects of cisplatin on mitochondrial dynamics and function. Larvae treated with 10mug/ml cisplatin had normal survival with deficiencies in righting and heat sensing behavior. Behavior was abrogated by, the pan caspase inhibitor, p35. However, active caspase 3 was not detected by immunostaining. There was a 27% decrease in mitochondrial membrane potential and a 42% increase in reactive oxygen species (ROS) in mitochondria along the axon. Examination of mitochondrial axonal trafficking showed no changes in velocity, flux or mitochondrial length. However, cisplatin treatment resulted in a greater number of stationary organelles caused by extended pausing during axonal motility. These results demonstrate that cisplatin induces behavior deficiencies in Drosophila larvae, decreased mitochondrial activity, increased ROS production and mitochondrial pausing without killing the larvae. Thus, this study identified particular aspects of mitochondrial dynamics and function that are affected in cisplatin-induced peripheral neuropathy and may represent key therapeutic targets.
Sofola-Adesakin, O., Khericha, M., Snoeren, I., Tsuda, L. and Partridge, L. (2016). pGluAβ increases accumulation of Aβ in vivo and exacerbates its toxicity. Acta Neuropathol Commun 4: 109. PubMed ID: 27717375
Several species of β-amyloid peptides (&Abeta;; see Drosophila Appl) exist as a result of differential cleavage from amyloid precursor protein (APP) to yield various C-terminal Aβ peptides. Several N-terminal modified Aβ peptides have also been identified in Alzheimer's disease (AD) brains, the most common of which is pyroglutamate-modified Aβ (AβpE3-42). AβpE3-42 peptide has an increased propensity to aggregate, appears to accumulate in the brain before the appearance of clinical symptoms of AD, and precedes Aβ1-42 deposition. Moreover, in vitro studies have shown that AβpE3-42 can act as a seed for full length Aβ1-42. This study characterized the Drosophila model of AβpE3-42 toxicity by expressing the peptide in specific sets of neurons using the GAL4-UAS system, and measuring different phenotypic outcomes. AβpE3-42 peptide was found to have an increased propensity to aggregate. Expression of AβpE3-42 in the neurons of adult flies led to behavioural dysfunction and shortened lifespan. Expression of AβpE3-42 constitutively in the eyes led to disorganised ommatidia, and activation of the c-Jun N-terminal kinase (JNK) signaling pathway. The eye disruption was almost completely rescued by co-expressing a candidate Aβ degrading enzyme, neprilysin2. Furthermore, neprilysin2 was capable of degrading AβpE3-42. Also, the seeding hypothesis was tested for AβpE3-42 in vivo, and its effect on Aβ1-42 levels were measured. Aβ1-42 levels were significantly increased when Aβ1-42 and AβpE3-42 peptides were co-expressed. Furthermore, AβpE3-42 enhanced Aβ1-42 toxicity in vivo. These findings implicate AβpE3-42 as an important source of toxicity in AD, and suggest that its specific degradation could be therapeutic.
Song, Q., Feng, G., Huang, Z., Chen, X., Chen, Z. and Ping, Y. (2016). Aberrant axonal arborization of PDF neurons induced by Aβ42-mediated JNK activation underlies sleep disturbance in an Alzheimer's model. Mol Neurobiol [Epub ahead of print]. PubMed ID: 27718103
Impaired sleep patterns are common symptoms of Alzheimer's disease (AD). Cellular mechanisms underlying sleep disturbance in AD remain largely unknown. Here, using a Drosophila Aβ42 AD model, Aβ42 was shown to markedly decrease sleep in a large population, accompanied with postdevelopmental axonal arborization of wake-promoting pigment-dispersing factor (PDF) neurons. The arborization is mediated in part via JNK activation and can be reversed by decreasing JNK signaling activity. Axonal arborization and impaired sleep are correlated in Aβ42 and JNK kinase hemipterous mutant flies. Image reconstruction revealed that these aberrant fibers preferentially project to pars intercerebralis (PI), a fly brain region analogous to the mammalian hypothalamus. Moreover, PDF signaling in PI neurons was found to modulate sleep/wake activities, suggesting that excessive release of PDF by these aberrant fibers may lead to the impaired sleep in Aβ42 flies. Finally, inhibition of JNK activation in Aβ42 flies restores nighttime sleep loss, decreases Aβ42 accumulation, and attenuates neurodegeneration. These data provide a new mechanism by which sleep disturbance could be induced by Aβ42 burden, a key initiator of a complex pathogenic cascade in AD.
Vanhoutte, D., Schips, T. G., Kwong, J. Q., Davis, J., Tjondrokoesoemo, A., Brody, M. J., Sargent, M. A., Kanisicak, O., Yi, H., Gao, Q. Q., Rabinowitz, J. E., Volk, T., McNally, E. M. and Molkentin, J. D. (2016). Thrombospondin expression in myofibers stabilizes muscle membranes. Elife 5 [Epub ahead of print]. PubMed ID: 27669143
Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. This study shows that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of βPS integrin. This functional conservation emphasizes the fundamental importance of Thbs as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy.

Saturday, October 12th

Towler, B. P., Jones, C. I., Harper, K. L., Waldron, J. A. and Newbury, S. F. (2016). A novel role for the 3'-5' exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth. RNA Biol [Epub ahead of print]. PubMed ID: 27630034
This study has shown that the exoribonuclease Dis3L2 is required for regulation of proliferation in the wing imaginal discs in Drosophila. Dis3L2 is a member of a highly conserved family of exoribonucleases that degrade RNA in a 3'-5' direction. Knockdown of Dis3L2 results in substantial wing overgrowth due to increased cellular proliferation rather than an increase in cell size. Imaginal discs are specified in the embryo before proliferating and differentiating to form the adult structures of the fly. Using RNA-seq, a small set of mRNAs was identified that are sensitive to Dis3L2 activity. Of the mRNAs which increase in levels and are therefore potential targets of Dis3L2, two were identified that change at the post-transcriptional level but not at the transcriptional level, namely CG2678 (a transcription factor) and pyrexia (a TRP cation channel). A compensatory effect between Dis3L2 and the 5'-3' exoribonuclease Pacman was identified, demonstrating that these two exoribonucleases function to regulate opposing pathways within the developing tissue. This work provides the first description of the molecular and developmental consequences of Dis3L2 inactivation in a non-human animal model. The work is directly relevant to the understanding of human overgrowth syndromes such as Perlman syndrome.
Reimao-Pinto, M. M., Manzenreither, R. A., Burkard, T. R., Sledz, P., Jinek, M., Mechtler, K. and Ameres, S. L. (2016). Molecular basis for cytoplasmic RNA surveillance by uridylation-triggered decay in Drosophila. EMBO J [Epub ahead of print]. PubMed ID: 27729457
The posttranscriptional addition of nucleotides to the 3' end of RNA regulates the maturation, function, and stability of RNA species in all domains of life. This study shows that in flies, 3' terminal RNA uridylation triggers the processive, 3'-to-5' exoribonucleolytic decay via the RNase II/R enzyme CG16940, a homolog of the human Perlman syndrome exoribonuclease Dis3l2. Together with the TUTase Tailor, dmDis3l2 forms the cytoplasmic, terminal RNA uridylation-mediated processing (TRUMP) complex that functionally cooperates in the degradation of structured RNA RNA immunoprecipitation and high-throughput sequencing reveals a variety of TRUMP complex substrates, including abundant non-coding RNA, such as 5S rRNA, tRNA, snRNA, snoRNA, and the essential RNase MRP. Based on genetic and biochemical evidence, a key function is proposed of the TRUMP complex in the cytoplasmic quality control of RNA polymerase III transcripts. Together with high-throughput biochemical characterization of dmDis3l2 and bacterial RNase R, these results imply a conserved molecular function of RNase II/R enzymes as "readers" of destabilizing posttranscriptional marks - uridylation in eukaryotes and adenylation in prokaryotes - that play important roles in RNA surveillance.
Smylla, T.K., Preiss, A. and Maier, D. (2016). In vivo analysis of internal ribosome entry at the Hairless locus by genome engineering in Drosophila. Sci Rep 6: 34881. PubMed ID: 27713501
Cell communication in metazoans requires the Notch signaling pathway, which is subjected to strict regulation of both activation and silencing. In Drosophila, silencing involves the assembly of a repressor complex by Hairless (H) on Notch target gene promoters. An in-frame internal ribosome entry site in the full length H transcript results in two H protein isoforms (Hp120 and Hp150). Hence, H may repress Notch signalling activity in situations where cap-dependent translation is inhibited. This study demonstrates the in vivo importance of both H isoforms for proper fly development. To this end, the endogenous H locus was replaced by constructs specifically affecting translation of either Hp150 or Hp120 isoforms using genome engineering. Findings indicate the functional relevance of both H proteins. Based on bristle phenotypes, the predominant isoform Hp150 appears to be of particular importance. In contrast, growth regulation and venation of the wing require the concomitant activity of both isoforms. Finally, the IRES dependent production of Hp120 during mitosis was verified in vivo. Together these data confirm IRES mediated translation of H protein in vivo, supporting strict regulation of Notch in different cellular settings.

Li, Y., Li, S., Jin, P., Chen, L. and Ma, F. (2016). miR-11 regulates pupal size of Drosophila melanogaster via directly targeting Ras85D. Am J Physiol Cell Physiol: ajpcell 00190 02016. PubMed ID: 27733364
MicroRNAs play diverse roles in various physiological processes during Drosophila development. This study reports that miR-11 regulates pupal size during Drosophila metamorphosis via targeting Ras85D with following evidences: pupal size was increased in the miR-11 deletion mutant; restoration of miR-11 in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant; ectopic expression of miR-11 in brain insulin-producing cells (IPCs) and whole body shows consistent alteration of pupal size; Dilps and Ras85D expressions were negatively regulated by miR-11 in vivo; miR-11 targets Ras85D through directly binding to Ras85D 3'UTR in vitro; removal of one copy of Ras85D in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant. Thus, this work provides a novel mechanism of pupal size determination by microRNAs during Drosophila melanogaster metamorphosis.

Friday, November 11th

Wei, Y., Reveal, B., Cai, W. and Lilly, M.A. (2016). The GATOR1 complex regulates metabolic homeostasis and the response to nutrient stress in Drosophila melanogaster. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27672113
TORC1 regulates metabolism and growth in response to a large array of upstream inputs. The evolutionary conserved trimeric GATOR1 complex inhibits TORC1 activity in response to amino acid limitation. In humans, the GATOR1 complex has been implicated in a wide array of pathologies including cancer and hereditary forms of epilepsy. However, the precise role of GATOR1 in animal physiology remains largely undefined. This study characterized null mutants of the GATOR1 components nprl2, nprl3 and iml1 in Drosophila melanogaster. It was demonstrated that all three mutants have inappropriately high baseline levels of TORC1 activity and decreased adult viability. Consistent with increased TORC1 activity, GATOR1 mutants exhibit a cell autonomous increase in cell growth. Notably, escaper nprl2 and nprl3 mutant adults have a profound locomotion defect. In line with a non-autonomous role in the regulation of systemic metabolism, expressing the Nprl3 protein in the fat body, a nutrient storage organ, and hemocytes but not muscles and neurons rescues the motility of nprl3 mutants. Finally, nprl2 and nprl3 mutants fail to activate autophagy in response to amino acid limitation and are extremely sensitive to both amino acid and complete starvation. Thus, in Drosophila, in addition to maintaining baseline levels of TORC1 activity, the GATOR1 complex has retained a critical role in the response to nutrient stress. In summary, the TORC1 inhibitor GATOR1 contributes to multiple aspects of the development and physiology of Drosophila.

Williams, C. M., McCue, M. D., Sunny, N. E., Szejner-Sigal, A., Morgan, T. J., Allison, D. B. and Hahn, D. A. (2016). Cold adaptation increases rates of nutrient flow and metabolic plasticity during cold exposure in Drosophila melanogaster. Proc Biol Sci 283. PubMed ID: 27605506
Metabolic flexibility is an important component of adaptation to stressful environments, including thermal stress and latitudinal adaptation. The direct relationship between selection on thermal stress hardiness and metabolic flux has not previously been tested. This study investigated flexibility of nutrient catabolism during cold stress in Drosophila artificially selected for fast or slow recovery from chill coma (i.e. cold-hardy or -susceptible), specifically testing the hypothesis that stress adaptation increases metabolic turnover. Using 13C-labelled glucose, this study first showed that cold-hardy flies more rapidly incorporate ingested carbon into amino acids and newly synthesized glucose, permitting rapid synthesis of proline, a compound shown elsewhere to improve survival of cold stress. Second, using glucose and leucine tracers cold-hardy flies were shown to have higher oxidation rates than cold-susceptible flies before cold exposure, similar oxidation rates during cold exposure, and returned to higher oxidation rates during recovery. Additionally, cold-hardy flies transferred compounds among body pools more rapidly during cold exposure and recovery. Increased metabolic turnover may allow cold-adapted flies to better prepare for, resist and repair/tolerate cold damage. This work illustrates for the first time differences in nutrient fluxes associated with cold adaptation, suggesting that metabolic costs associated with cold hardiness could invoke resource-based trade-offs that shape life histories.
Wang, C. W., Purkayastha, A., Jones, K. T., Thaker, S. K. and Banerjee, U. (2016). In vivo genetic dissection of tumor growth and the Warburg effect. Elife 5 [Epub ahead of print]. PubMed ID: 27585295
A well-characterized metabolic landmark for aggressive cancers is the reprogramming from oxidative phosphorylation to aerobic glycolysis, referred to as the Warburg effect. Models mimicking this process are often incomplete due to genetic complexities of tumors and cell lines containing unmapped collaborating mutations. In order to establish a system where individual components of oncogenic signals and metabolic pathways can be readily elucidated, this study induced a glycolytic tumor in the Drosophila wing imaginal disc by activating the oncogene PDGF/VEGF-receptor (Pvr). This causes activation of multiple oncogenic pathways including Ras, PI3K/Akt, Raf/ERK, Src and JNK. Together this network of genes stabilizes Hifα (Sima) that in turn, transcriptionally up-regulates many genes encoding glycolytic enzymes. Collectively, this network of genes also causes inhibition of pyruvate dehydrogenase (PDH) activity resulting in diminished ox-phos levels. The high ROS produced during this process functions as a feedback signal to consolidate this metabolic reprogramming.
Gruntenko, N. E., et al. (2016). The impact of FOXO on dopamine and octopamine metabolism in Drosophila under normal and heat stress conditions. Biol Open. PubMed ID: 27754851
FOXO is a component of the insulin signalling pathway and plays a role in responding to adverse conditions, such as oxidative stress and starvation. In stressful conditions, FOXO moves from the cytosol to the nucleus where it activates gene expression programmes. This study shows that FOXO in Drosophila melanogaster responds to heat stress as it does to other stressors. The catecholamine signalling pathway is another component of the stress response. In Drosophila, dopamine and octopamine levels rise steeply under heat, nutrition and mechanical stresses, which is followed by a decrease in the activity of synthesis enzymes. This study demonstrates that the nearly twofold decline of FOXO expression in foxoBG01018 mutants results in dramatic changes in the metabolism of dopamine and octopamine and the overall response to stress. The absence of FOXO increases tyrosine decarboxylase activity, the first enzyme in octopamine synthesis, and decreases the enzymatic activity of enzymes in dopamine synthesis, alkaline phosphatase and tyrosine hydroxylase, in young Drosophila females. This study identified the juvenile hormone as a mediator of FOXO regulation of catecholamine metabolism. These findings suggest that FOXO is a possible trigger for endocrinological stress reactions.

Thursday, November 10th

Lee, S. H., Kim, Y. J. and Choi, S. Y. (2016). BMP signaling modulates the probability of neurotransmitter release and readily releasable pools in Drosophila neuromuscular junction synapses. Biochem Biophys Res Commun 479: 440-446. PubMed ID: 27671198
The structure and function of synapses is modulated by the interaction of presynaptic and postsynaptic neurons via cell adhesion molecules or secreted signal molecules. Bone morphogenic protein (BMP) is a secreted molecule mediating retrograde signaling that is involved in the formation and maintenance of synaptic structure throughout many animal species. However, how BMP signaling modulates presynaptic neurotransmitter release is not yet clear. This study examined the function of BMP signaling factors in neurotransmitter release in Drosophila neuromuscular synapses using loss-of-function mutants in genes for BMP modulators, Wit, Mad, and Dad. Larvae with mutations in wit and mad commonly showed a decreased synaptic bouton number in neuromuscular synapses. Larvae with dad mutations showed an increased bouton number. The amplitudes of miniature EJC (mEJC) were normal for these mutants. wit and mad mutants showed decreased evoked EJC (eEJC) amplitude and increased paired pulse facilitation, implying impaired presynaptic neurotransmitter release. A reduction was found in readily releasable neurotransmitters pool sizes in wit and mad mutants. However, dad mutants showed a normal probability of neurotransmitter release and readily releasable pool sizes and normal eEJC amplitude even with clear abnormalities in synaptic structure. These results suggested that BMP signaling is critical for each step of presynaptic neurotransmission. The results also suggested that BMP signaling modulates both synaptic structure and function independently and specifically.
Gupta, V. K., Pech, U., Bhukel, A., Fulterer, A., Ender, A., Mauermann, S. F., Andlauer, T. F., Antwi-Adjei, E., Beuschel, C., Thriene, K., Maglione, M., Quentin, C., Bushow, R., Schwarzel, M., Mielke, T., Madeo, F., Dengjel, J., Fiala, A. and Sigrist, S. J. (2016). Spermidine suppresses age-associated memory impairment by preventing adverse increase of presynaptic active zone size and release. PLoS Biol 14: e1002563. PubMed ID: 27684064
Memories are assumed to be formed by sets of synapses changing their structural or functional performance. The efficacy of forming new memories declines with advancing age, but the synaptic changes underlying age-induced memory impairment remain poorly understood. Spermidine feeding has been found to specifically suppress age-dependent impairments in forming olfactory memories, providing a mean to search for synaptic changes involved in age-dependent memory impairment. This study shows that a specific synaptic compartment, the presynaptic active zone (AZ) of the adult brain, increases the size of its ultrastructural elaboration and releases significantly more synaptic vesicles with advancing age. These age-induced AZ changes, however, were fully suppressed by spermidine feeding. A genetically enforced enlargement of AZ scaffolds (four gene-copies of BRP) impaired memory formation in young animals. Thus, in the Drosophila nervous system, aging AZs seem to steer towards the upper limit of their operational range, limiting synaptic plasticity and contributing to impairment of memory formation. Spermidine feeding suppresses age-dependent memory impairment by counteracting these age-dependent changes directly at the synapse. The results suggest that the integrity of the autophagic system is crucial for the spermidine-mediated protection from age-associated increase in AZ scaffold components
Choubey, P. K. and Roy, J. K. (2016). Rab11, a vesicular trafficking protein, affects endoreplication through Ras-mediated pathway in Drosophila melanogaster. Cell Tissue Res [Epub ahead of print]. PubMed ID: 27677270
Rab11, a small monomeric GTPase associated with recycling endosomes, is a key molecule in the regulation of vesicular trafficking and is involved in the development and differentiation of many Drosophila tissues through interaction with diverse signaling pathways. This study reportsthat Rab11 affects endoreplication through a Ras-mediated pathway. Suppression of Rab11 activity in salivary glands, an endoreplicating tissue, leads to reduction in size of salivary glands with cells having a small nucleus. Endoreplication-regulating proteins, CycE, E2f1 and Gem, are also down-regulated in Rab11 knocked-down salivary glands suggesting that Rab11 has a role in the process of endoreplication, possibly indirectly through other pathways that regulate cell cycle progression. Ras signaling plays an important role in cell cycle progression through G/S phase transition. Ectopic expression of activated Ras in salivary glands of Rab11 down-regulated individuals rescues the small-sized glands to intermediate size. Furthermore, altered localization of Ras was observed in Rab11 down-regulated salivary glands. It is likely that the low level of endoreplication in the Rab11 down-regulated condition is Ras-mediated.
Walkup, W. G., Mastro, T. L., Schenker, L. T., Vielmetter, J., Hu, R., Iancu, A., Reghunathan, M., Bannon, B. D. and Kennedy, M. B. (2016). A model for regulation by SynGAP-alpha1 of binding of synaptic proteins to PDZ-domain 'Slots' in the postsynaptic density. Elife 5 [Epub ahead of print]. PubMed ID: 27623146 Evolutionary Homolog Study
SynGAP is a Ras/Rap GTPase-activating protein (GAP) that is a major constituent of postsynaptic densities (PSDs) from mammalian forebrain. Its α1 isoform binds to all three PDZ (PSD-95, Discs-large, ZO-1) domains of PSD-95 (see Drosophila Discs large), the principal PSD scaffold, and can occupy as many as 15% of these PDZ domains. Evidence is presented that synGAP-α1 regulates the composition of the PSD by restricting binding to the PDZ domains of PSD-95. Phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII; see Drosophila CaMKII) and Polo-like kinase-2 (PLK2; see Drosophila Polo) decreases its affinity for the PDZ domains by several fold, which would free PDZ domains for occupancy by other proteins. Finally, three critical postsynaptic signaling proteins that bind to the PDZ domains of PSD-95 are shown to be present in higher concentration in PSDs isolated from mice with a heterozygous deletion of synGAP.

Wednesday, November 10th

Melkov, A., Baskar, R., Alcalay, Y. and Abdu, U. (2016). New mode of mitochondrial transport and polarized sorting regulation by Dynein, Milton and Miro. Development [Epub ahead of print]. PubMed ID: 27707795
Intrinsic cell microtubule (MT) polarity, together with molecular motors and adaptor proteins, determines mitochondrial polarized targeting and MT-dependent transport. In polarized cells, such as neurons, mitochondrialmobility and transport require the regulation of kinesin and dynein by two adaptor proteins, Milton and Miro. Recent, studies have found that Dynein heavy chain 64C (Dhc64C) is the primary motor protein for both anterograde and retrograde transport of mitochondria in the Drosophila bristle. This study revealed that a molecular lesion in the Dhc64C allele that reduced bristle mitochondrial velocity generated a variant that acts as a "slow" dynein in a MT gliding assay, indicative of dynein directly regulating mitochondrial transport. It was also shown that in milton RNAi flies, mitochondrial flux into the bristle shaft but not velocity was significantly reduced. Surprisingly, mitochondria retrograde flux but not net velocity was significantly decreased in miro RNAi flies. This study thus revealed a new mode of mitochondrial polarized sorting in polarized cell growth, whereby bi-directional mitochondrial transport undertaken exclusively by dynein is regulated by Milton in anterograde direction and by a Miro-dependent switch to retrograde direction.
Pratt, M. B., Titlow, J. S., Davis, I., Barker, A. R., Dawe, H. R., Raff, J. W. and Roque, H. (2016). Drosophila sensory cilia lacking MKS-proteins exhibit striking defects in development but only subtle defects in adults. J Cell Sci. PubMed ID: 27577095
Cilia are conserved organelles that have important motility, sensory and signalling roles. The transition zone (TZ) at the base of the cilium is critical for cilia function, and defects in several TZ proteins are associated with human congenital ciliopathies such as Nephronophthisis (NPHP) and Meckel Gruber syndrome (MKS: see MKS1, Tectonic, B9D1 and B9D2). In several species, MKS and NPHP proteins form separate complexes that cooperate with Cep290 to assemble the TZ, but flies appear to lack core components of the NPHP module. This study shows that MKS proteins in flies are spatially separated from Cep290 at the TZ, and that flies mutant for individual MKS genes fail to recruit other MKS proteins to the TZ, while Cep290 appears to be recruited normally. Although there are abnormalities in microtubule and membrane organisation in developing MKS mutant cilia, these defects are less apparent in adults, where sensory cilia and sperm flagella appear to function quite normally. Thus, localising MKS proteins to the cilium or flagellum is not essential for viability or fertility in flies.
Ducuing, A. and Vincent, S. (2016). The actin cable is dispensable in directing dorsal closure dynamics but neutralizes mechanical stress to prevent scarring in the Drosophila embryo. Nat Cell Biol [Epub ahead of print]. PubMed ID: 27749820
The actin cable is a supracellular structure that embryonic epithelia produce to close gaps. However, the action of the cable remains debated. This study has addressed the function of the cable using Drosophila dorsal closure, a paradigm to understand wound healing. First, the actin cytoskeleton scaffold protein Zasp52 was shown to be specifically required for actin cable formation. Next, Zasp52 loss of function to dissect the mechanism of action of the cable. Surprisingly, closure dynamics are perfect in Zasp52 mutants: the cable is therefore dispensable for closure, even in the absence of the amnioserosa. Conversely, it was observed that the cable protects cellular geometries from robust morphogenetic forces that otherwise interfere with closure: the absence of cable results in defects in epithelial organization that lead to morphogenetic scarring. It is proposed that the cable prevents morphogenetic scarring by stabilizing cellular interactions rather than by acting on closure dynamics.
Otani, T., Ogura, Y., Misaki, K., Maeda, T., Kimpara, A., Yonemura, S. and Hayashi, S. (2016). IKK inhibits PKC to promote Fascin-dependent actin bundling. Development [Epub ahead of print]. PubMed ID: 27578797

Signaling molecules have pleiotropic functions and are activated by various extracellular stimuli. Protein kinase C (PKC) is activated by diverse receptors, and its dysregulation is associated with diseases including cancer. However, how the undesired activation of PKC is prevented during development remains poorly understood. Previous studies have shown that a protein kinase, IKK, is active at the growing bristle tip and regulates actin bundle organization during Drosophila bristle morphogenesis. This study demonstrated that IKK regulates the actin bundle localization of a dynamic actin cross-linker, Fascin. IKK inhibits PKC, thereby protecting Fascin from its inhibitory phosphorylation. Excess PKC activation is responsible for the actin bundle defects in ikk-deficient bristles, whereas PKC is dispensable for bristle morphogenesis in wildtype bristles, indicating that PKC is repressed by IKK in wildtype bristle cells. These results suggest that IKK prevents excess activation of PKC during bristle morphogenesis.

Tuesday, November 8th

Villa, R., Schauer, T., Smialowski, P., Straub, T. and Becker, P. B. (2016). PionX sites mark the X chromosome for dosage compensation. Nature 537: 244-248. PubMed ID: 27580037
The rules defining which small fraction of related DNA sequences can be selectively bound by a transcription factor are poorly understood. One of the most challenging tasks in DNA recognition is posed by dosage compensation systems that require the distinction between sex chromosomes and autosomes. In Drosophila melanogaster, the male-specific lethal dosage compensation complex (MSL-DCC) doubles the level of transcription from the single male X chromosome, but the nature of this selectivity is not known. Previous efforts to identify X-chromosome-specific target sequences were unsuccessful as the identified MSL recognition elements lacked discriminative power. Therefore, additional determinants such as co-factors, chromatin features, RNA and chromosome conformation have been proposed to refine targeting further. Using an in vitro genome-wide DNA binding assay this study shows that recognition of the X chromosome is an intrinsic feature of the MSL-DCC. MSL2, the male-specific organizer of the complex, uses two distinct DNA interaction surfaces-the CXC and proline/basic-residue-rich domains-to identify complex DNA elements on the X chromosome. Specificity is provided by the CXC domain, which binds a novel motif defined by DNA sequence and shape. This motif characterizes a subclass of MSL2-binding sites, which has been named PionX (pioneering sites on the X) as they appeared early during the recent evolution of an X chromosome in D. miranda and are the first chromosomal sites to be bound during de novo MSL-DCC assembly. These data provide the first documented molecular mechanism through which the dosage compensation machinery distinguishes the X chromosome from an autosome. They highlight fundamental principles in the recognition of complex DNA elements by protein that will have a strong impact on many aspects of chromosome biology.
Avva, S. V. and Hart, C. M. (2016). Characterization of the Drosophila BEAF-32A and BEAF-32B insulator proteins. PLoS One 11: e0162906. PubMed ID: 27622635
Data implicate the Drosophila 32 kDa Boundary Element-Associated Factors BEAF-32A and BEAF-32B in both chromatin domain insulator element function and promoter function. They might also function as an epigenetic memory by remaining bound to mitotic chromosomes. Both proteins are made from the same gene. They differ in their N-terminal 80 amino acids, which contain single DNA-binding BED fingers. The remaining 200 amino acids are identical in the two proteins. The structure and function of the middle region of 120 amino acids is unknown, while the C-terminal region of 80 amino acids has a putative leucine zipper and a BESS domain and mediates BEAF-BEAF interactions. This study reports a further characterization of BEAF. The BESS domain alone is shown to be sufficient to mediate BEAF-BEAF interactions, although the presence of the putative leucine zipper on at least one protein strengthens the interactions. BEAF-32B is sufficient to rescue a null BEAF mutation in flies. Using mutant BEAF-32B rescue transgenes, the middle region and the BESS domain was shown to be essential. In contrast, the last 40 amino acids of the middle region, which is poorly conserved among Drosophila species, is dispensable. Deleting the putative leucine zipper results in a hypomorphic mutant BEAF-32B protein. Finally, the dynamics are documented of BEAF-32A-EGFP and BEAF-32B-mRFP during mitosis in embryos. A subpopulation of both proteins appears to remain on mitotic chromosomes and also on the mitotic spindle, while much of the fluorescence is dispersed during mitosis. Differences in the dynamics of the two proteins are observed in syncytial embryos, and both proteins show differences between syncytial and later embryos. This characterization of BEAF lays a foundation for future studies into molecular mechanisms of BEAF function.
Xie, W., Chojnowski, A., Boudier, T., Lim, J. S., Ahmed, S., Ser, Z., Stewart, C. and Burke, B. (2016). A-type lamins form distinct filamentous networks with differential nuclear pore complex associations. Curr Biol. PubMed ID: 27641764
Evolutionary Homolog Study
The nuclear lamina is a universal feature of metazoan nuclear envelopes (NEs) [1]. In mammalian cells, it appears as a 10-30 nm filamentous layer at the nuclear face of the inner nuclear membrane (INM) and is composed primarily of A- and B-type lamins, members of the intermediate filament family. While providing structural integrity to the NE, the lamina also represents an important signaling and regulatory platform. Two A-type lamin isoforms, lamins A and C (LaA and LaC; see Drosophila Lamin), are expressed in most adult human cells. Encoded by a single gene, these proteins are largely identical, diverging only in their C-terminal tail domains. By contrast with that of LaC, the unique LaA tail undergoes extensive processing, including farnesylation and endo-proteolysis. However, functional differences between LaA and LaC are still unclear. Compounding this uncertainty, the structure of the lamina remains ill defined. In this study, BioID, an in vivo proximity-labeling method was used to identify differential interactors of A-type lamins. One of these, Tpr, a nuclear pore complex (NPC) protein, is highlighted by its selective association with LaC. By employing superresolution microscopy, this Tpr association was demonstrated to be mirrored in enhanced interaction of LaC with NPCs. Further superresolution studies visualizing both endogenous A- and B-type lamins have allowed construction of a nanometer-scale model of the mammalian nuclear lamina. These data indicate that different A- and B-type lamin species assemble into separate filament networks that together form an extended composite structure at the nuclear periphery providing attachment sites for NPCs, thereby regulating their distribution.
Zhang, Z., English, B. P., Grimm, J. B., Kazane, S. A., Hu, W., Tsai, A., Inouye, C., You, C., Piehler, J., Schultz, P. G., Lavis, L. D., Revyakin, A. and Tjian, R. (2016). Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis. Genes Dev 30: 2106-2118. PubMed ID: 27798851
Evolutionary Homolog Study
Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II; see Drosophila Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, this study established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Real-time measurements were performed of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB (see Drosophila RNA polymerase and general transcription factors). Surprisingly, it was found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB-promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II-TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

Monday, November 7th

Cichewicz, K., Garren, E. J., Adiele, C., Aso, Y., Wang, Z., Wu, M., Birman, S., Rubin, G. M. and Hirsh, J. (2016). A new brain dopamine deficient Drosophila and its pharmacological and genetic rescue. Genes Brain Behav [Epub ahead of print]. PubMed ID: 27762066
Drosophila tyrosine hydroxylase (DTH) is the rate limiting enzyme for Dopamine (DA) biosynthesis. Viable brain DA deficient flies were previously generated using tissue selective GAL4-UAS binary expression rescue of a DTH null mutation and these flies show specific behavioral impairments. To circumvent the limitations of rescue via binary expression, this study achieved rescue utilizing genomically integrated mutant DTH. As expected, DA deficient flies have no detectable DTH or DA in the brain, and show reduced locomotor activity. This deficit can be rescued by L-DOPA/carbidopa feeding, similar to human Parkinson's disease treatment. Genetic rescue via GAL4/UAS-DTH was also successful, although this required the generation of a new UAS-DTH1 transgene devoid of most untranslated regions, since existing UAS-DTH transgenes express in the brain without a Gal4 driver via endogenous regulatory elements. A surprising finding of the newly constructed UAS-DTH1m is that it expresses DTH at an undetectable level when regulated by dopaminergic GAL4 drivers even when fully rescuing DA, indicating that DTH immunostaining is not necessarily a valid marker for DA expression. This finding necessitated optimizing DA immunohistochemistry, revealing details of DA innervation to the mushroom body and the central complex. When DA rescue is limited to specific DA neurons, DA does not diffuse beyond the DTH-expressing terminals, such that DA signaling can be limited to very specific brain regions.
Bernardo-Garcia, F. J., Humberg, T. H., Fritsch, C. and Sprecher, S. G. (2016). Successive requirement of Glass and Hazy for photoreceptor specification and maintenance in Drosophila. Fly (Austin): [Epub ahead of print]. PubMed ID: 27723419
Development of the insect compound eye requires a highly controlled interplay between transcription factors. However, the genetic mechanisms that link early eye field specification to photoreceptor terminal differentiation and fate maintenance remain largely unknown. This study deciphered the function of two transcription factors, Glass and Hazy, which play a central role during photoreceptor development. The regulatory interactions between Glass and Hazy suggest that they function together in a coherent feed-forward loop in all types of Drosophila photoreceptors. While the glass mutant eye lacks the expression of virtually all photoreceptor genes, young hazy mutants correctly express most phototransduction genes. Interestingly, the expression of these genes is drastically reduced in old hazy mutants. This age-dependent loss of the phototransduction cascade correlates with a loss of phototaxis in old hazy mutant flies. It is concluded that Glass can either directly or indirectly initiate the expression of most phototransduction proteins in a Hazy-independent manner, and that Hazy is mainly required for the maintenance of functional photoreceptors in adult flies.
Raccuglia, D., Yan McCurdy, L., Demir, M., Gorur-Shandilya, S., Kunst, M., Emonet, T. and Nitabach, M. N. (2016). Presynaptic GABA receptors mediate temporal contrast enhancement in Drosophila olfactory sensory neurons and modulate odor-driven behavioral kinetics. eNeuro 3 [Epub ahead of print]. PubMed ID: 27588305
Contrast enhancement mediated by lateral inhibition within the nervous system enhances the detection of salient features of visual and auditory stimuli, such as spatial and temporal edges. However, it remains unclear how mechanisms for temporal contrast enhancement in the olfactory system can enhance the detection of odor plume edges during navigation. To address this question, pulses of high odor intensity that induce sustained peripheral responses in olfactory sensory neurons (OSNs) were delivered to Drosophila melanogaster flies. Optical electrophysiology was used to directly measure electrical responses in presynaptic terminals, and it was demonstrated that sustained peripheral responses are temporally sharpened by the combined activity of two types of inhibitory GABA receptors, ionotropic GABAA and metabotropic GABAB inhibitory receptors to generate contrast-enhanced voltage responses in central OSN axon terminals. Furthermore, it was shown how these GABA receptors modulate the time course of innate behavioral responses after odor pulse termination, demonstrating an important role for temporal contrast enhancement in odor-guided navigation.
Akin, O. and Zipursky, S. L. (2016). Frazzled promotes growth cone attachment at the source of a Netrin gradient in the Drosophila visual system. Elife 5 [Epub ahead of print]. PubMed ID: 27743477
Axon guidance is proposed to act through a combination of long- and short-range attractive and repulsive cues. The ligand-receptor pair, Netrin (Net) and Frazzled (Fra) (DCC, Deleted in Colorectal Cancer, in vertebrates), is recognized as the prototypical effector of chemoattraction, with roles in both long- and short-range guidance. In the Drosophila visual system, R8 photoreceptor growth cones were shown to require Net-Fra to reach their target, the peak of a Net gradient. Using live imaging, it was shown, however, that R8 growth cones reach and recognize their target without Net, Fra, or Trim9, a conserved binding partner of Fra, but do not remain attached to it. Thus, despite the graded ligand distribution along the guidance path, Net-Fra is not used for chemoattraction. Based on findings in other systems, it is proposed that adhesion to substrate-bound Net underlies both long- and short-range Net-Fra-dependent guidance in vivo, thereby eroding the distinction between them.

Sunday, November 6th

Johnson, J. L., Huang, W., Roman, G. and Costa-Mattioli, M. (2015). TORC2: a novel target for treating age-associated memory impairment. Sci Rep 5: 15193. PubMed ID: 26489398
Memory decline is one of the greatest health threats of the twenty-first century. Because of the widespread increase in life expectancy, 20 percent of the global population will be over 60 in 2050 and the problems caused by age-related memory loss will be dramatically aggravated. However, the molecular mechanisms underlying this inevitable process are not well understood. This study shows that the activity of the recently discovered mechanistic target of rapamycin (mTOR) complex 2 (mTORC2, see Drosophila TOR and Rictor) declines with age in the brain of both fruit flies and rodents and that the loss of mTORC2-mediated actin polymerization contributes to age-associated memory loss. Intriguingly, treatment with a small molecule that activates mTORC2 (A-443654; a specific Akt inhibitor that activates mTORC2-mediated phosphorylation of Akt) reverses long-term memory (LTM) deficits in both aged mice and flies. In addition, pharmacologically boosting either mTORC2 or actin polymerization enhances LTM. In contrast to the current approaches to enhance memory that have primarily targeted the regulation of gene expression (epigenetic, transcriptional, and translational), the data points to a novel, evolutionarily conserved mechanism for restoring memory that is dependent on structural plasticity. These insights into the molecular basis of age-related memory loss may hold promise for new treatments for cognitive disorders.
Fischer, B., Luthy, K., Paesmans, J., De Koninck, C., Maes, I., Swerts, J., Kuenen, S., Uytterhoeven, V., Verstreken, P. and Versees, W. (2016). Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function. Nat Struct Mol Biol. PubMed ID: 27669036
Mutations in TBC1D24 cause severe epilepsy and DOORS syndrome, but the molecular mechanisms underlying these pathologies are unresolved. This study solved the crystal structure of the TBC domain of the Drosophila ortholog Skywalker, revealing an unanticipated cationic pocket conserved among TBC1D24 homologs. Cocrystallization and biochemistry showed that this pocket binds phosphoinositides phosphorylated at the 4 and 5 positions. The most prevalent patient mutations affect the phosphoinositide-binding pocket and inhibit lipid binding. Using in vivo photobleaching of Skywalker-GFP mutants, including pathogenic mutants, it was shown that membrane binding via this pocket restricts Skywalker diffusion in presynaptic terminals. Additionally, the pathogenic mutations cause severe neurological defects in flies, including impaired synaptic-vesicle trafficking and seizures, and these defects are reversed by genetically increasing synaptic PI(4,5)P2 concentrations through synaptojanin mutations. Hence, this study has discovered that a TBC domain affected by clinical mutations directly binds phosphoinositides through a cationic pocket and that phosphoinositide binding is critical for presynaptic function.
Lee, K. H., et al. (2016). C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles. Cell 167: 774-788 e717. PubMed ID: 27768896
Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. This study identified the interactome of all DPRs and found that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, it was shown that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.
Kramer, N. J., et al. (2016). Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts. Science 353: 708-712. PubMed ID: 27516603
An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. This study found that targeting the transcription elongation factor Spt4 (see Drosophila Spt4) selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. In Drosophila, Spt4 RNAi partially suppressed the degenerative phenotype of the external and internal eye in (GGGGCC)49-expressing flies and almost completely suppressed the retinal thinning normally observed in (GGGGCC)29-expressing flies. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately.

Saturday, November 5th

Alton, L. A., Condon, C., White, C. R. and Angilletta, M. J. (2016). Colder environments did not select for a faster metabolism during experimental evolution of Drosophila melanogaster. Evolution [Epub ahead of print]. PubMed ID: 27757954
The effect of temperature on the evolution of metabolism has been the subject of debate for a century; however, no consistent patterns have emerged from comparisons of metabolic rate within and among species living at different temperatures. This study used experimental evolution to determine how metabolism evolves in populations of Drosophila melanogaster exposed to one of three selective treatments: a constant 16 ° C, a constant 25 ° C, or temporal fluctuations between 16 and 25 ° C. August Krogh's controversial hypothesis was tested that colder environments select for a faster metabolism. Given that colder environments also experience greater seasonality, the hypothesis was also tested that temporal variation in temperature may be the factor that selects for a faster metabolism. The metabolic rate of flies from each selective treatment was measured at 16, 20.5, and 25 ° C. Although metabolism was faster at higher temperatures, flies from the selective treatments had similar metabolic rates at each measurement temperature. Based on variation among genotypes within populations, heritable variation in metabolism was likely sufficient for adaptation to occur. It is concluded that colder or seasonal environments do not necessarily select for a faster metabolism. Rather, other factors besides temperature likely contribute to patterns of metabolic rate over thermal clines in nature.
Dalla, S. and Dobler, S. (2016). Gene duplications circumvent trade-offs in enzyme function: Insect adaptation to toxic host plants. Evolution [Epub ahead of print]. PubMed ID: 27683239
Evolutionary Homolog Study
Herbivorous insects and their adaptations against plant toxins provide striking opportunities to investigate the genetic basis of traits involved in coevolutionary interactions. Target site insensitivity to cardenolides has evolved convergently across six orders of insects, involving identical substitutions in the Na,K-ATPase gene and repeated convergent gene duplications. The large milkweed bug, Oncopeltus fasciatus, has three copies of the Na,K-ATPase alpha-subunit gene that bear differing numbers of amino acid substitutions in the binding pocket for cardenolides. To analyze the effect of these substitutions on cardenolide resistance and to infer possible trade-offs in gene function, the cardenolide-sensitive Na,K-ATPase of Drosophila melanogaster was expressed in vitro and four distinct combinations were introduced of substitutions observed in the three gene copies of O. fasciatus. With an increasing number of substitutions, the sensitivity of the Na,K-ATPase to a standard cardenolide decreased in a stepwise manner. At the same time, the enzyme's overall activity decreased significantly with increasing cardenolide resistance and only the least substituted mimic of the Na,K-ATPase α1C copy maintained activity similar to the wild-type enzyme. These results suggest that the Na,K-ATPase copies in O. fasciatus have diverged in function, enabling specific adaptations to dietary cardenolides while maintaining the functionality of this critical ion carrier.
Clifton, B. D., Librado, P., Yeh, S. D., Solares, E., Real, D., Jayasekera, S., Zhang, W., Shi, M., Park, R., Magie, R., Ma, H. C., Xia, X. Q., Marco, A., Rozas, J. and Ranz, J. M. (2016). Rapid functional and sequence differentiation of a tandemly-repeated species-specific multigene family in Drosophila. Mol Biol Evol [Epub ahead of print]. PubMed ID: 27702774
Gene clusters of recently duplicated genes are hotbeds for evolutionary change. However, understanding of how mutational mechanisms and evolutionary forces shape the structural and functional evolution of these clusters is hindered by the high sequence identity among the copies, which typically results in their inaccurate representation in genome assemblies. The presumed testis-specific, chimeric gene Sdic originated and tandemly expanded in Drosophila melanogaster, contributing to increased male-male competition. Using various types of massively parallel sequencing data, the organization, sequence evolution, and functional attributes of the different Sdic copies were examined. By leveraging long-read sequencing data, both copy number and order differences were uncovered from the currently accepted annotation for the Sdic region. Despite evidence for pervasive gene conversion affecting the Sdic copies, signatures of two episodes of diversifying selection were uncovered, that have contributed to the evolution of a variety of C-termini and miRNA binding site compositions. Expression analyses involving RNA-seq datasets from 59 different biological conditions revealed distinctive expression breadths among the copies, with three copies being transcribed in females, opening the possibility to a sexually antagonistic effect. Phenotypic assays using Sdic knock-out strains indicated that should this antagonistic effect exist, it does not compromise female fertility. These results strongly suggest that the genome consolidation of the Sdic gene cluster is more the result of a quick exploration of different paths of molecular tinkering by different copies than a mere dosage increase, which could be a recurrent evolutionary outcome in the presence of persistent sexual selection.
Assis, R. (2016). Transcriptional interference promotes rapid expression divergence of Drosophila nested genes. Genome Biol Evol [Epub ahead of print]. PubMed ID: 27664180
Nested genes are the most common form of protein-coding overlap in eukaryotic genomes. Previous studies have shown that nested genes accumulate rapidly over evolutionary time, typically via the insertion of short young duplicate genes into long introns. However, the evolutionary relationship between nested genes remains unclear. This study compare RNA-seq expression profiles of nested, proximal intra-chromosomal, intermediate intra-chromosomal, distant intra-chromosomal, and inter-chromosomal gene pairs in two Drosophila species. Expression profiles of nested genes were found to be more divergent than those of any other class of genes, supporting the hypothesis that concurrent expression of nested genes is deleterious due to transcriptional interference. Further analysis reveals that expression profiles of derived nested genes are more divergent than those of their ancestral un-nested orthologs, which are more divergent than those of un-nested genes with similar genomic features. Thus, gene expression divergence between nested genes is likely caused by selection against nesting of genes with insufficiently divergent expression profiles, as well as by continued expression divergence after nesting. Moreover, expression divergence and sequence evolutionary rates are elevated in young nested genes and reduced in old nested genes, indicating that a burst of rapid evolution occurs after nesting. Together, these findings suggest that similarity between expression profiles of nested genes is deleterious due to transcriptional interference, and that natural selection addresses this problem both by eradicating highly deleterious nestings and by enabling rapid expression divergence of surviving nested genes, thereby quickly limiting or abolishing transcriptional interference.

Friday, November 4th

Cho, E., Lee, E. and Kim, E. Y. (2016). Diversification of molecular clockwork for tissue specific function: insight from novel Drosophila Clock mutant homologous to mouse Clock allele. BMB Rep [Epub ahead of print]. PubMed ID: 27756446
The circadian clock system enables organisms to anticipate the rhythmic environmental changes and to manifest behavior and physiology at advantageous times of day. Transcriptional/translational feedback loop (TTFL) is the basic feature of eukaryotic circadian clock and is based on the rhythmic association of circadian transcriptional activator and repressor. In Drosophila, repression of dCLOCK/CYCLE (dCLK/CYC) mediated transcription by PERIOD (PER) is critical for inducing circadian rhythms of gene expression. Pacemaker neurons in the brain control specific circadian behaviors upon environmental timing cues such as light and temperature cycle. This study shows here that amino acids 657-707 of dCLK is important for the transcriptional activation and the association with PER both in vitro and in vivo. Flies expressing dCLK lacking AA657-707 in Clkout genetic background, homologous to the mouse Clock allele where exon 19 region is deleted, display pacemaker-neuron-dependent perturbation of the molecular clockwork. Namely, the molecular rhythms in light-cycle-sensitive pacemaker neurons such as ventral lateral neurons (LNvs) were significantly disrupted but those in temperature-cycle-sensitive pacemaker neurons such as dorsal neurons (DNs) were robust. These results suggest that the dCLK-controlled TTFL diversified in pacemaker-neuron-dependent manner which contribute to specific functions such as different sensitivities to entraining cues.
Berendes, V., Zill, S. N., Buschges, A. and Bockemuhl, T. (2016). Speed-dependent interplay between local pattern-generating activity and sensory signals during walking in Drosophila. J Exp Biol [Epub ahead of print]. PubMed ID: 27688052
In insects, the coordinated motor output required for walking is based on the interaction between local pattern-generating networks providing basic rhythmicity and leg sensory signals which modulate this output on a cycle-to-cycle basis. How this interplay changes speed-dependently and thereby gives rise to the different coordination patterns observed at different speeds is understood insufficiently. This study used amputation to reduce sensory signals in single legs and decouple them mechanically during walking in Drosophila. This allowed for the dissociation between locally-generated motor output in the stump and coordinating influences from intact legs. Leg stumps were still rhythmically active during walking. While the oscillatory frequency in intact legs was dependent on walking speed, stumps showed a high and relatively constant oscillation frequency at all walking speeds. At low walking speeds there was no strict cycle-to-cycle coupling between stumps and intact legs. In contrast, at high walking speeds stump oscillations were strongly coupled to the movement of intact legs on a 1-to-1 basis. While during slow walking there was no preferred phase between stumps and intact legs, a preferred time interval was found between touch-down or lift-off events in intact legs and levation or depression of stumps. Based on these findings, it is hypothesized that, as in other insects, walking speed in Drosophila is predominantly controlled by indirect mechanisms and that direct modulation of basic pattern-generating circuits plays a subsidiary role. Furthermore, inter-leg coordination strength seems to be speed-dependent and greater coordination is evident at higher walking speeds.
Berman, G. J., Bialek, W. and Shaevitz, J. W. (2016). Predictability and hierarchy in Drosophila behavior. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27702892
Even the simplest of animals exhibit behavioral sequences with complex temporal dynamics. Prominent among the proposed organizing principles for these dynamics has been the idea of a hierarchy, wherein the movements an animal makes can be understood as a set of nested subclusters. Although this type of organization holds potential advantages in terms of motion control and neural circuitry, measurements demonstrating this for an animal's entire behavioral repertoire have been limited in scope and temporal complexity. This study use a recently developed unsupervised technique to discover and track the occurrence of all stereotyped behaviors, including locomotion and grooming, performed by fruit flies moving in a shallow arena. Calculating the optimally predictive representation of the fly's future behaviors, it was shown that fly behavior exhibits multiple time scales and is organized into a hierarchical structure that is indicative of its underlying behavioral programs and its changing internal states.
Du, E. J., Ahn, T. J., Wen, X., Seo, D. W., Na, D. L., Kwon, J. Y., Choi, M., Kim, H. W., Cho, H. and Kang, K. (2016). Nucleophile sensitivity of Drosophila TRPA1 underlies light-induced feeding deterrence. Elife 5 [Epub ahead of print]. PubMed ID: 27656903
Solar irradiation including ultraviolet (UV) light causes tissue damage by generating reactive free radicals that can be electrophilic or nucleophilic due to unpaired electrons. Little is known about how free radicals induced by natural sunlight are rapidly detected and avoided by animals. This study discovered that Drosophila Transient Receptor Potential Ankyrin 1 (TRPA1), previously known only as an electrophile receptor, sensitively detects photochemically active sunlight through nucleophile sensitivity. Rapid light-dependent feeding deterrence in Drosophila was mediated only by the TRPA1A isoform, despite the TRPA1A and TRPA1B isoforms having similar electrophile sensitivities. Such isoform dependence re-emerges in the detection of structurally varied nucleophilic compounds and nucleophilicity-accompanying hydrogen peroxide (H2O2). Furthermore, these isoform-dependent mechanisms require a common set of TRPA1A-specific residues dispensable for electrophile detection. Collectively, TRPA1A rapidly responds to natural sunlight intensities through its nucleophile sensitivity as a receptor of photochemically generated radicals, leading to an acute light-induced behavioral shift in Drosophila.

Thursday, November 3rd

Tamori, Y., Suzuki, E. and Deng, W. M. (2016). Epithelial tumors originate in tumor hotspots, a tissue-intrinsic microenvironment. PLoS Biol 14: e1002537. PubMed ID: 27584724
Malignant tumors are caused by uncontrolled proliferation of transformed mutant cells that have lost the ability to maintain tissue integrity. Although a number of causative genetic backgrounds for tumor development have been discovered, the initial steps mutant cells take to escape tissue integrity and trigger tumorigenesis remain elusive. This study shows through analysis of conserved neoplastic tumor-suppressor genes (nTSGs) in Drosophila wing imaginal disc epithelia that tumor initiation depends on tissue-intrinsic local cytoarchitectures, causing tumors to consistently originate in a specific region of the tissue. In this "tumor hotspot" where cells constitute a network of robust structures on their basal side, nTSG-deficient cells delaminate from the apical side of the epithelium and begin tumorigenic overgrowth by exploiting endogenous JAK/STAT signaling activity. Conversely, in other regions, the "tumor coldspot" nTSG-deficient cells are extruded toward the basal side and undergo apoptosis. When the direction of delamination is reversed through suppression of RhoGEF2, an activator of the Rho family small GTPases, and JAK/STAT is activated ectopically in these coldspot nTSG-deficient cells, tumorigenesis is induced. These data indicate that two independent processes, apical delamination and JAK/STAT activation, are concurrently required for the initiation of nTSG-deficient-induced tumorigenesis. Given the conservation of the epithelial cytoarchitecture, tumorigenesis may be generally initiated from tumor hotspots by a similar mechanism.
Lin, Z., Guo, H., Cao, Y., Zohrabian, S., Zhou, P., Ma, Q., VanDusen, N., Guo, Y., Zhang, J., Stevens, S.M., Liang, F., Quan, Q., van Gorp, P.R., Li, A., Dos Remedios, C., He, A., Bezzerides, V.J. and Pu, W.T. (2016). Acetylation of VGLL4 regulates Hippo-YAP signaling and postnatal cardiac growth. Dev Cell [Epub ahead of print]. PubMed ID: 27720608
Evolutionary Homolog Study:
Binding of the transcriptional co-activator YAP (see Drosophila yki) with the transcription factor TEAD (see Drosophila sd) stimulates growth of the heart (see dorsal vessel in Drosophila) and other organs. YAP overexpression potently stimulates fetal cardiomyocyte (CM) proliferation, but YAP's mitogenic potency declines postnatally. While investigating factors that limit YAP's postnatal mitogenic activity, this study found that the CM-enriched TEAD1 binding protein VGLL4 (see Drosophila CG1737) inhibits CM proliferation by inhibiting TEAD1-YAP interaction and by targeting TEAD1 for degradation. Importantly, VGLL4 acetylation at lysine 225 negatively regulated its binding to TEAD1. This developmentally regulated acetylation event critically governs postnatal heart growth, since overexpression of an acetylation-refractory VGLL4 mutant enhanced TEAD1 degradation, limits neonatal CM proliferation, and causes CM necrosis. These data define an acetylation-mediated, VGLL4-dependent switch that regulates TEAD stability and YAP-TEAD activity. These insights may improve targeted modulation of TEAD-YAP activity in applications from cardiac regeneration to cancer.

Verghese, S. and Su, T. T. (2016). Drosophila Wnt and STAT define apoptosis-resistant epithelial cells for tissue regeneration after irradiation. PLoS Biol 14: e1002536. PubMed ID: 27584613
Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR) that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. This study has identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. These findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue.
Wang, S., Lu, Y., Yin, M. X., Wang, C., Wu, W., Li, J., Wu, W., Ge, L., Hu, L., Zhao, Y. and Zhang, L. (2016). Importin α1 mediates Yorkie nuclear import via an N-terminal non-canonical nuclear localization signal. J Biol Chem 291: 7926-7937. PubMed ID: 26887950
The Hippo signaling pathway controls organ size by orchestrating cell proliferation and apoptosis. When the Hippo pathway is inactivated, the transcriptional co-activator Yorkie translocates into the nucleus and forms a complex with transcription factor Scalloped to promote the expression of Hippo pathway target genes. Therefore, the nuclear translocation of Yorkie is a critical step in Hippo signaling. This study provides evidence that the N-terminal 1-55 amino acids of Yorkie, especially Arg-15, were essential for its nuclear localization. By mass spectrometry and biochemical analyses, it was found that Importin α1 can directly interact with the Yorkie N terminus and drive Yorkie into the nucleus. Further experiments show that the upstream component Hippo can inhibit Importin α1-mediated Yorkie nuclear import. Taken together, this study has identified a potential nuclear localization signal at the N-terminal end of Yorkie as well as a critical role for Importin alpha1 in Yorkie nuclear import.

Wednesday, November 2nd

Nesler, K. R., Starke, E. L., Boin, N. G., Ritz, M. and Barbee, S. A. (2016). Presynaptic CamKII regulates activity-dependent axon terminal growth. Mol Cell Neurosci 76: 33-41. PubMed ID: 27567686
Spaced synaptic depolarization induces rapid axon terminal growth and the formation of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). This study identified a novel presynaptic function for the Calcium/Calmodulin-dependent Kinase II (CamKII) protein in the control of activity-dependent synaptic growth. Consistent with this function, both total and phosphorylated CamKII (p-CamKII) are were found to be enriched in axon terminals. Interestingly, p-CamKII appears to be enriched at the presynaptic axon terminal membrane. Moreover, levels of total CamKII protein within presynaptic boutons globally increase within one hour following stimulation. These effects correlate with the activity-dependent formation of new presynaptic boutons. The increase in presynaptic CamKII levels is inhibited by treatment with cyclohexamide suggesting a protein-synthesis dependent mechanism. Previous work has found that acute spaced stimulation rapidly downregulates levels of neuronal microRNAs (miRNAs) that are required for the control of activity-dependent axon terminal growth at this synapse. The rapid activity-dependent accumulation of CamKII protein within axon terminals is inhibited by overexpression of activity-regulated miR-289 in motor neurons. Experiments in vitro using a CamKII translational reporter show that miR-289 can directly repress the translation of CamKII via a sequence motif found within the CamKII 3' untranslated region (UTR). Collectively, these studies support the idea that presynaptic CamKII acts downstream of synaptic stimulation and the miRNA pathway to control rapid activity-dependent changes in synapse structure.
Haugas, M., Tikker, L., Achim, K., Salminen, M. and Partanen, J. (2016). Gata2 and Gata3 regulate the differentiation of serotonergic and glutamatergic neuron subtypes of the dorsal raphe. Development [Epub ahead of print]. PubMed ID: 27789623
Evolutionary Homolog Study
Serotonergic and glutamatergic neurons of the dorsal raphe regulate many brain functions and are important for mental health. Their functional diversity is based on molecularly distinct subtypes; however, the development of this heterogeneity is poorly understood. This study shows that the ventral neuroepithelium of mouse anterior hindbrain is divided into specific subdomains giving rise to serotonergic neurons as well as other types of neurons and glia. The newly born serotonergic precursors are segregated into distinct subpopulations expressing vesicular glutamate transporter 3 (Vglut3) or serotonin transporter (Sert). These populations differ in their requirements for transcription factors Gata2 and Gata3 (see Drosophila Serpent), activated in the post-mitotic precursors. Gata2 operates upstream of Gata3 as a cell fate selector in both populations, whereas Gata3 is important for the differentiation of the Sert+ precursors and for the serotonergic identity of the Vglut3+ precursors. Similar to the serotonergic neurons, the Vglut3 expressing glutamatergic neurons, located in the central dorsal raphe, are derived from neural progenitors in the ventral hindbrain and express Pet1. Furthermore, both Gata2 and Gata3 are redundantly required for their differentiation. This study demonstrates lineage relationships of the dorsal raphe neurons and suggests that functionally significant heterogeneity of these neurons is established early during their differentiation.
Chakraborty, S., Deb, B. K., Chorna, T., Konieczny, V., Taylor, C. W. and Hasan, G. (2016). Mutant IP3 receptors attenuate store-operated Ca2+ entry by destabilizing STIM-Orai interactions in Drosophila neurons. J Cell Sci [Epub ahead of print]. PubMed ID: 27591258
Store-operated Ca2+ entry (SOCE) occurs when loss of Ca2+ from the endoplasmic reticulum (ER) stimulates the Ca2+ sensor, STIM, to cluster and activate the plasma membrane (PM) Ca2+ channel, Orai. Inositol 1,4,5-trisphosphate receptors (IP3R) are assumed to regulate SOCE solely by mediating ER Ca2+ release. This study shows that in Drosophila neurons, mutant IP3R attenuate SOCE evoked by depleting Ca2+ stores with thapsigargin. In normal neurons, store depletion caused STIM and IP3R to accumulate near the PM, association of STIM with Orai, clustering of STIM and Orai at ER-PM junctions, and activation of SOCE. These responses were attenuated in neurons with mutant IP3R and rescued by over-expression of STIM with Orai. It is concluded that after depletion of Ca2+ stores in Drosophila, translocation of IP3R to ER-PM junctions facilitates the coupling of STIM to Orai that leads to activation of SOCE (Chakraborty, 2016).
Gupta, T., Kumar, A., Pierre, C., VijayRaghavan, K. and Giangrande, A. (2016). The Glide/Gcm fate determinant controls initiation of collective cell migration by regulating Frazzled. Elife 5 [Epub ahead of print]. PubMed ID: 27740455
Collective migration is a complex process that contributes to build precise tissue and organ architecture. Several molecules involved in cell interaction control collective migration, but what their precise role is and how is their expression finely tuned to orchestrate the different steps of the process is poorly understood. This study shows that the timely and threshold expression of the Netrin receptor Frazzled triggers the initiation of glia migration in the Drosophila wing. Frazzled expression is induced by the Glide/Gcm transcription factor in a dose dependent manner. Thus, the glial determinant also regulates the efficiency of collective migration. NetrinB but not NetrinA serves as a chemoattractant and Unc5 contributes as a repellant Netrin receptor for glia migration. This model includes strict spatial localization of a ligand, a cell autonomously acting receptor and a fate determinant that act coordinately to direct glia towards their final destination.

Tuesday, November 1st

Stanishneva-Konovalova, T. B., Kelley, C. F., Eskin, T. L., Messelaar, E. M., Wasserman, S. A., Sokolova, O. S. and Rodal, A. A. (2016). Coordinated autoinhibition of F-BAR domain membrane binding and WASp activation by Nervous Wreck. Proc Natl Acad Sci U S A 113: E5552-5561. PubMed ID: 27601635
Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. This study used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott-Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. These results define structural rearrangements in Nwk that control F-BAR-membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo.
Lu, Z., Chouhan, A. K., Borycz, J. A., Lu, Z., Rossano, A. J., Brain, K. L., Zhou, Y., Meinertzhagen, I. A. and Macleod, G. T. (2016). High-probability neurotransmitter release sites represent an energy-efficient design. Curr Biol 26: 2562-2571. PubMed ID: 27593375
Nerve terminals contain multiple sites specialized for the release of neurotransmitters. This study tested the hypothesis that high-probability release sites represent an energy-efficient design. Release site probabilities and energy efficiency were examined at the terminals of two glutamatergic motor neurons synapsing on the same muscle fiber in Drosophila larvae. Through electrophysiological and ultrastructural measurements, calculated release site probabilities were found to differ considerably between terminals (0.33 versus 0.11). The energy required to release and recycle glutamate were calculated from the same measurements. The energy required to remove calcium and sodium ions subsequent to nerve excitation was estimated through microfluorimetric and morphological measurements. Energy efficiency was calculated as the number of glutamate molecules released per ATP molecule hydrolyzed, and high-probability release site terminals were found to be more efficient (0.13 versus 0.06). This analytical model indicates that energy efficiency is optimal (~ 0.15) at high release site probabilities (~0.76). As limitations in energy supply constrain neural function, high-probability release sites might ameliorate such constraints by demanding less energy. Energy efficiency can be viewed as one aspect of nerve terminal function, in balance with others, because high-efficiency terminals depress significantly during episodic bursts of activity.
Wang, M., Chen, P. Y., Wang, C. H., Lai, T. T., Tsai, P. I., Cheng, Y. J., Kao, H. H. and Chien, C. T. (2016). Dbo/Henji modulates synaptic dPAK to gate glutamate receptor abundance and postsynaptic response. PLoS Genet 12: e1006362. PubMed ID: 27736876
In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. This study analyzed mutant synapses of henji, also known as dbo, at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but that of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. It is proposed that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK.
Kennedy, A. J., Rahn, E. J., Paulukaitis, B. S., Savell, K. E., Kordasiewicz, H. B., Wang, J., Lewis, J. W., Posey, J., Strange, S. K., Guzman-Karlsson, M. C., Phillips, S. E., Decker, K., Motley, S. T., Swayze, E. E., Ecker, D. J., Michael, T. P., Day, J. J. and Sweatt, J. D. (2016). Tcf4 regulates synaptic plasticity, DNA methylation, and memory function. Cell Rep 16: 2666-2685. PubMed ID: 27568567
Evolutionary Homolog Study
Human haploinsufficiency of the bHLH transcription factor Tcf4 (see Drosophila Daughterless) leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS), which is associated with severe language impairment and development delay. This study demonstrates that Tcf4 haploinsufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4+/- mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, small-molecule HDAC inhibitors were found to normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4-deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified "memory-associated" genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, it was observed that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4+/- mice.

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