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Monday, June 30th, 2014

Ciglar, L., Girardot, C., Wilczynski, B., Braun, M. and Furlong, E. E. (2014). Coordinated repression and activation of two transcriptional programs stabilizes cell fate during myogenesis. Development 141: 2633-2643. PubMed ID: 24961800
Summary: Molecular models of cell fate specification typically focus on the activation of specific lineage programs. However, the concurrent repression of unwanted transcriptional networks is also essential to stabilize certain cellular identities, as shown in a number of diverse systems and phyla. This study demonstrates that this dual requirement also holds true in the context of Drosophila myogenesis. By integrating genetics and genomics, a new role for the pleiotropic transcriptional repressor Tramtrack69 was identified in myoblast specification. Drosophila muscles are formed through the fusion of two discrete cell types: founder cells (FCs) and fusion-competent myoblasts (FCMs). When tramtrack69 is removed, FCMs appear to adopt an alternative muscle FC-like fate. Conversely, ectopic expression of this repressor phenocopies muscle defects seen in loss-of-function lame duck mutants, a transcription factor specific to FCMs. This occurs through Tramtrack69-mediated repression in FCMs, whereas Lame duck activates a largely distinct transcriptional program in the same cells. Lineage-specific factors are therefore not sufficient to maintain FCM identity. Instead, their identity appears more plastic, requiring the combination of instructive repressive and activating programs to stabilize cell fate.

Kiritooshi, N., Yorimitsu, T., Shirai, T., Puli, O. R., Singh, A. and Nakagoshi, H. (2014). A vertex specific dorsal selector Dve represses the ventral appendage identity in Drosophila head. Mech Dev [Epub ahead of print]. PubMed ID: 24971779
Summary: Developmental fields are subdivided into lineage-restricted cell populations, known as compartments. In the eye imaginal disc of Drosophila, dorso-ventral (DV) lineage restriction is the primary event, whereas antero-posterior compartment boundary is the first lineage restriction in other imaginal discs. The Iroquois complex (Iro-C) genes function as dorsal selectors and repress the default, ventral, identity in the eye-head primordium. In Iro-C mutant clones, change of the dorsal identity to default ventral fate leads to generation of ectopic DV boundary, which results in dorsal eye enlargement, and duplication of ventral appendages like antenna and maxillary palp. Similar phenotypes were observed in heads with defective proventriculus (dve) mutant clones. This study shows that the homeobox gene dve is a downstream effector of Iro-C in the dorsal head capsule (vertex) specification and represses the ventral (antennal) identity. Two homeodomain proteins Distal-less (Dll) and Homothorax (Hth) are known to be determinants of the antennal identity. Ectopic antenna formation in heads with dve mutant clones was associated with ectopic Dll expression and endogenous Hth expression in the vertex region. Interestingly, dveDll double mutant clones could also induce ectopic antennae lacking the distal structures, suggesting that the Dve activity is crucial for repressing inappropriate antenna-forming potential in the vertex region. These results clearly indicate that not only the activation of effector genes to execute developmental program but also the repression of inappropriate program is crucial for establishment of the organ identity.

Khan, Z., Wang, Y. C., Wieschaus, E. F. and Kaschube, M. (2014). Quantitative 4D analyses of epithelial folding during Drosophila gastrulation. Development [Epub ahead of print]. PubMed ID: 24948599
Summary: Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. This study reports the development of EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. The results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during ventral furrow formation, a complex epithelial folding event in the early Drosophila embryo. This tool should be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts.

Zulueta-Coarasa, T., Tamada, M., Lee, E. J. and Fernandez-Gonzalez, R. (2014). Automated multidimensional image analysis reveals a role for Abl in embryonic wound repair. Development [Epub ahead of print]. PubMed ID: 24948602
Summary: The embryonic epidermis displays a remarkable ability to repair wounds rapidly. Embryonic wound repair is driven by the evolutionary conserved redistribution of cytoskeletal and junctional proteins around the wound. Drosophila has emerged as a model to screen for factors implicated in wound closure. However, genetic screens have been limited by the use of manual analysis methods. This study introduces MEDUSA, a novel image-analysis tool for the automated quantification of multicellular and molecular dynamics from time-lapse confocal microscopy data. MEDUSA was validated by quantifying wound closure in Drosophila embryos, and the results of the automated analysis are comparable to analysis by manual delineation and tracking of the wounds, while significantly reducing the processing time. MEDUSA can also be applied to the investigation of cellular behaviors in three and four dimensions. Using MEDUSA, it was found that the conserved nonreceptor tyrosine kinase Abelson (Abl) contributes to rapid embryonic wound closure. Abl plays a role in the organization of filamentous actin and the redistribution of the junctional protein beta-catenin at the wound margin during embryonic wound repair. Finally, different models for the role of Abl in the regulation of actin architecture and adhesion dynamics at the wound margin are discussed.

Sunday, June 29th

Goto, A., Fukuyama, H., Imler, J. L. and Hoffmann, J. A. (2014). The chromatin regulator DMAP1 modulates activity of the NF-kappaB transcription factor Relish in the Drosophila innate immune response. J Biol Chem [Epub ahead of print]. PubMed ID: 24947515
Summary: The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-kappaB family, the Toll and the IMD pathways. The latter shares extensive similarities with the mammalian TNF-R pathway and was initially discovered for its role in anti-Gram-negative bacterial reactions. A previous interactome study reported that an unexpectedly large number of proteins are binding to the canonical components of the IMD pathway. This study focussed on DNA methyltransferase associated protein 1 (DMAP1), which this study identified as an interactant of Relish, a Drosophila transcription factor reminiscent of the mammalian p105 NF-kappaB protein. Silencing of DMAP1 expression both in S2 cells and in flies results in a significant reduction of E. coli induced expression of antimicrobial peptides. Epistatic analysis indicates that DMAP1 acts in parallel or downstream of Relish. Co-immunoprecipitation experiments further reveal that, in addition to Relish, DMAP1 also interacts with Akirin and the Brahma associated protein 55 kDa (Bap55). Taken together, these results reveal that DMAP1 is a novel nuclear modulator of the IMD pathway, possibly acting at the level of chromatin remodeling.

Varma, D., Bulow, M. H., Pesch, Y. Y., Loch, G. and Hoch, M. (2014). Forkhead, a new cross regulator of metabolism and innate immunity downstream of TOR in Drosophila. J Insect Physiol. PubMed ID: 24842780
Summary: Antimicrobial peptides (AMPs) are conserved cationic peptides which both act as defense molecules of the host immune system and as regulators of the commensal microbiome. Expression of AMPs is induced in response to infection by the Toll and Imd pathway. Under non-infected conditions, the transcription factor dFOXO directly regulates a set of AMP expression at low levels when nutrients are limited. This study has analyzed whether Target of rapamycin (TOR), another major regulator of growth and metabolism, also modulates AMP responses in Drosophila. Downregulation of TOR by feeding the drug rapamycin or by overexpressing the negative TOR regulators TSC1/TSC2 was shown to result in a specific induction of the AMPs Diptericin (Dpt) and Metchnikowin (Mtk). In contrast, overexpression of Rheb, which positively regulates TOR led to a repression of the two AMPs. Genetic and pharmacological experiments indicate that Dpt and Mtk activation is controlled by the transcription factor Forkhead (Fkh), the founding member of the FoxO family. Shuttling of Fkh from the cytoplasm to the nucleus is induced in the fat body and in the posterior midgut in response to TOR downregulation. The Fkh-dependent induction of Dpt and Mtk can be triggered in dFOXO null mutants and in immune-compromised Toll and IMD pathway mutants indicating that FKH acts in parallel to these regulators. Together, this study has discovered that FKH is the second conserved member of the FoxO family cross-regulating metabolism and innate immunity. dFOXO and FKH, which are activated upon downregulation of insulin or TOR activities, respectively, act in parallel to induce different sets of AMPs, thereby modulating the immune status of metabolic tissues such as the fat body or the gut in response to the oscillating energy status of the organism.

Horn, L., Leips, J. and Starz-Gaiano, M. (2014). Phagocytic ability declines with age in adult Drosophila hemocytes. Aging Cell [Epub ahead of print]. PubMed ID: 24828474
Summary: Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. This study examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age-related immune dysfunction. The localized response was characterized of immune cells at the dorsal vessel to bacterial infection in 1-week- and 5-week-old flies. A quantitative phagocytosis assay was developed for adult Drosophila, and this was used to characterize the effect of age on phagocytosis in transgenic and natural variant lines. Genes necessary for bacterial engulfment in other contexts are also required in adult flies. Blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. These results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.

Mallon, E. B., Alghamdi, A., Holdbrook, R. T. and Rosato, E. (2014). Immune stimulation reduces sleep and memory ability in Drosophila melanogaster. PeerJ 2: e434. PubMed ID: 24949247
Summary: Psychoneuroimmunology studies the increasing number of connections between neurobiology, immunology and behaviour. This study demonstrates the effects of the immune response on two fundamental behaviours: sleep and memory ability in Drosophila melanogaster. The Geneswitch system was used to upregulate peptidoglycan receptor protein (PGRP) expression, thereby stimulating the immune system in the absence of infection. Geneswitch was activated by feeding the steroid RU486, to the flies. An aversive classical conditioning paradigm was used to quantify memory and measures of activity to infer sleep. Immune stimulated flies exhibited reduced levels of sleep, which could not be explained by a generalised increase in waking activity. Immune stimulated flies also showed a reduction in memory abilities. These results lend support to Drosophila as a model for immune-neural interactions and provide a possible role for sleep in the interplay between the immune response and memory.

Saturday, June 28th

Petsalaki, E. and Zachos, G. (2014). Chk2 prevents mitotic exit when the majority of kinetochores are unattached. J Cell Biol 205: 339-356. PubMed ID: 24798733
Summary: The spindle checkpoint delays exit from mitosis in cells with spindle defects. This paper shows that Chk2 is required to delay anaphase onset when microtubules are completely depolymerized but not in the presence of relatively few unattached kinetochores. Mitotic exit in Chk2-deficient cells correlates with reduced levels of Mono polar spindle 1 (Mps1) protein and increased Cdk1-tyrosine 15 inhibitory phosphorylation. Chk2 localizes to kinetochores and is also required for Aurora B-serine 331 phosphorylation in nocodazole or unperturbed early prometaphase. Serine 331 phosphorylation contributes to prometaphase accumulation in nocodazole after partial Mps1 inhibition and is required for spindle checkpoint establishment at the beginning of mitosis. In addition, expression of a phosphomimetic S331E mutant Aurora B rescues chromosome alignment or segregation in Chk2-deficient cells. It is proposed that Chk2 stabilizes Mps1 and phosphorylates Aurora B-serine 331 to prevent mitotic exit when most kinetochores are unattached. These results highlight mechanisms of an essential function of Chk2 in mitosis.

Nam, H. J. and van Deursen, J. M. (2014). Cyclin B2 and p53 control proper timing of centrosome separation. Nat Cell Biol 16: 538-549. PubMed ID: 24776885
Summary: Cyclins B1 and B2 (see Drosophila Cyclin B) are frequently elevated in human cancers and are associated with tumour aggressiveness and poor clinical outcome; however, whether and how B-type cyclins drive tumorigenesis is unknown. This study shows that cyclin B1 and B2 transgenic mice are highly prone to tumours, including tumour types where B-type cyclins serve as prognosticators. Cyclins B1 and B2 both induce aneuploidy when overexpressed but through distinct mechanisms, with cyclin B1 inhibiting separase activation, leading to anaphase bridges, and cyclin B2 triggering aurora-A-mediated Plk1 hyperactivation (see Drosophila aurora and polo), resulting in accelerated centrosome separation and lagging chromosomes. Complementary experiments revealed that cyclin B2 and p53 (see Drosophila p53) act antagonistically to control aurora-A-mediated centrosome splitting and accurate chromosome segregation in normal cells. These data demonstrate a causative link between B-type cyclin overexpression and tumour pathophysiology, and uncover previously unknown functions of cyclin B2 and p53 in centrosome separation that may be perturbed in many human cancers.

Collins, K. A., Unruh, J. R., Slaughter, B. D., Yu, Z., Lake, C. M., Nielsen, R. J., Box, K. S., Miller, D. E., Blumenstiel, J. P., Perera, A. G., Malanowski, K. E. and Hawley, R. S. (2014). Corolla Is a Novel Protein that Contributes to the Architecture of the Synaptonemal Complex of Drosophila. Genetics [Epub ahead of print]. PubMed ID: 24913682
Summary: In most organisms the synaptonemal complex (SC) connects paired homologs along their entire length during much of meiotic prophase. To better understand the structure of the SC, this study aimed to identify its components and determine how each of these components contributes to SC function. A novel SC component has been identified in Drosophila melanogaster female oocytes, which was named Corolla (CG8316). Using structured illumination microscopy, it was demonstrated that Corolla is a component of the central region of the SC. Consistent with its localization, yeast two-hybrid analysis showed that Corolla strongly interacts with Corona (Cona), a central element protein, demonstrating the first direct interaction between two inner-synaptonemal complex proteins in Drosophila. These observations help provide a more complete model of SC structure and function in Drosophila females.

Friday, June 27th

Biteau, B. and Jasper, H. (2014). Slit/Robo Signaling Regulates Cell Fate Decisions in the Intestinal Stem Cell Lineage of Drosophila. Cell Rep [Epub ahead of print]. PubMed ID: 24931602
Summary: In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. This study has identified a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. EEs were found to secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, it was shown that this lineage decision is made within ISCs and requires induction of the transcription factor Prospero in ISCs. This work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. The results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage.

Shimamura, M., Kyotani, A., Azuma, Y., Yoshida, H., Binh Nguyen, T., Mizuta, I., Yoshida, T., Mizuno, T., Nakagawa, M., Tokuda, T. and Yamaguchi, M. (2014). Genetic link between Cabeza, a Drosophila homologue of fused in sarcoma (FUS), and the EGFR signaling pathway. Exp Cell Res [Epub ahead of print]. PubMed ID: 24928275
Summary: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila Cabeza (Caz) is a FUS homologue; specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror, suppresses the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescues the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila.

Kim, K., Vinayagam, A. and Perrimon, N. (2014). A Rapid Genome-wide MicroRNA Screen Identifies miR-14 as a Modulator of Hedgehog Signaling. Cell Rep [Epub ahead of print]. PubMed ID: 24931604
Summary: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by binding to sequences within the 3' UTR of mRNAs. Because miRNAs bind to short sequences with partial complementarity, target identification is challenging. To complement the existing target prediction algorithms, a systematic 'reverse approach' screening platform was devised that allows the empirical prediction of miRNA-target interactions. Using Drosophila cells, the 3' untranslated regions (3' UTRs) of the Hedgehog pathway genes were screened against a genome-wide miRNA library, and both predicted and many nonpredicted miRNA-target interactions were identified. miR-14 was identified as essential for maintaining the proper level of Hedgehog signaling activity by regulating its physiological target, hedgehog. Furthermore, elevated levels of miR-14 suppress Hedgehog signaling activity by cotargeting its apparent nonphysiological targets, patched and smoothened. Altogether, this systematic screening platform is a powerful approach to identifying both physiological and apparent nonphysiological targets of miRNAs, which are relevant in both normal and diseased tissues.

Kane, L. A., Lazarou, M., Fogel, A. I., Li, Y., Yamano, K., Sarraf, S. A., Banerjee, S. and Youle, R. J. (2014). PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J Cell Biol 205: 143-153. PubMed ID: 24751536
Summary: PINK1 kinase (see Drosophila Pink1) activates the E3 ubiquitin ligase Parkin (see Drosophila Parkin) to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, it was discovered that endogenous PINK1 phosphorylates ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin's ubiquitin-like domain. Recombinant TcPINK1 from Tribolium directly phosphorylates ubiquitin, and phospho-ubiquitin activates Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibits Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.

Thursday, June 26th

Shimizu, H., Woodcock, S. A., Wilkin, M. B., Trubenova, B., Monk, N. A. and Baron, M. (2014). Compensatory Flux Changes within an Endocytic Trafficking Network Maintain Thermal Robustness of Notch Signaling. Cell 157: 1160-1174. PubMed ID: 24855951
Summary: Developmental signaling is remarkably robust to environmental variation, including temperature. For example, in ectothermic animals such as Drosophila, Notch signaling is maintained within functional limits across a wide temperature range. This study combined experimental and computational approaches to show that temperature compensation of Notch signaling is achieved by an unexpected variety of endocytic-dependent routes to Notch activation which, when superimposed on ligand-induced activation, act as a robustness module. Thermal compensation arises through an altered balance of fluxes within competing trafficking routes, coupled with temperature-dependent ubiquitination of Notch. This flexible ensemble of trafficking routes supports Notch signaling at low temperature but can be switched to restrain Notch signaling at high temperature and thus compensates for the inherent temperature sensitivity of ligand-induced activation. The outcome is to extend the physiological range over which normal development can occur. Similar mechanisms may provide thermal robustness for other developmental signals.

Iacobucci, G. J., Rahman, N. A., Valtuena, A. A., Nayak, T. K. and Gunawardena, S. (2014). Spatial and temporal characteristics of normal and perturbed vesicle transport. PLoS One 9: e97237. PubMed ID: 24878565
Summary: Efficient intracellular transport is essential for healthy cellular function and structural integrity, and problems in this pathway can lead to neuronal cell death and disease. To spatially and temporally evaluate how transport defects are initiated, this study adapted a primary neuronal culture system from Drosophila larval brains to visualize the movement dynamics of several cargos/organelles along a 90 micron axonal neurite over time. All six vesicles/organelles imaged showed robust bi-directional motility at both day 1 and day 2. Reduction of motor proteins decreased the movement of vesicles/organelles with increased numbers of neurite blocks. Neuronal growth was also perturbed with reduction of motor proteins. Strikingly, all blockages were not fixed, permanent blocks that impeded transport of vesicles as previously thought, but that some blocks were dynamic clusters of vesicles that resolved over time. Taken together, these findings suggest that non-resolving blocks may likely initiate deleterious pathways leading to death and degeneration, while resolving blocks may be benign. Therefore evaluating the spatial and temporal characteristics of vesicle transport has important implications for understanding of how transport defects can affect other pathways to initiate death and degeneration.

Pradhan-Sundd, T. and Verheyen, E. M. (2014). The role of Bro1- domain-containing protein Myopic in endosomal trafficking of Wnt/Wingless. Dev Biol [Epub ahead of print]. PubMed ID: 24821423
Summary: Wingless (Wg) proteins are secreted-lipid-modified glycoproteins involved in tissue patterning and cell-fate specification. Wg secretion is regulated by a specialized mechanism involving a repertoire of proteins including Wntless (Wls). This study shows that the Bro1-domain-containing protein Myopic (Mop) is indispensable for endosomal trafficking of Wg and Wls. Reductions in Mop leads to trapping of Wg and Wls in the early endosomes. Overexpression of the endosomal sorting protein Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) rescues the trafficking defect caused by the mop loss of function. The vertebrate homolog of Mop, Histidine domain-containing protein tyrosine phosphatase (HDPTP), was also found to have a conserved role in Wnt trafficking. These study highlights the importance of early endosomal trafficking for Wg secretion, and identifies a novel role for Mop in Wg signaling.

Faust, J. E., Manisundaram, A., Ivanova, P. T., Milne, S. B., Summerville, J. B., Brown, H. A., Wangler, M., Stern, M. and McNew, J. A. (2014). Peroxisomes Are Required for Lipid Metabolism and Muscle Function in Drosophila melanogaster. PLoS One 9: e100213. PubMed ID: 24945818
Summary: Peroxisomes are ubiquitous organelles that perform lipid and reactive oxygen species metabolism. Defects in peroxisome biogenesis cause peroxisome biogenesis disorders (PBDs). The most severe PBD, Zellweger syndrome, is characterized in part by neuronal dysfunction, craniofacial malformations, and low muscle tone (hypotonia). These devastating diseases lack effective therapies and the development of animal models may reveal new drug targets. Drosophila mutants with impaired peroxisome biogenesis were generated by disrupting the early peroxin gene Pex3, which participates in budding of pre-peroxisomes from the ER and peroxisomal membrane protein localization. Pex3 deletion mutants lack detectible peroxisomes and die before or during pupariation. At earlier stages of development, larvae lacking Pex3 display reduced size and impaired lipid metabolism. Selective loss of peroxisomes in muscles impairs muscle function and results in flightless animals. Although, hypotonia in PBD patients is thought to be a secondary effect of neuronal dysfunction, these results suggest that peroxisome loss directly affects muscle physiology, possibly by disrupting energy metabolism. Understanding the role of peroxisomes in Drosophila physiology, specifically in muscle cells may reveal novel aspects of PBD etiology.

Wednesday, June 25th

Guo, F., Cerullo, I., Chen, X. and Rosbash, M. (2014). PDF neuron firing phase-shifts key circadian activity neurons in Drosophila. Elife: e02780. PubMed ID: 24939987
Summary: Two long-standing models were addressed for the function of the Drosophila brain circadian network: a dual oscillator model, which emphasizes the primacy of PDF-containing neurons, and a cell-autonomous model for circadian phase adjustment. Five different circadian (E) neurons were identified that are a major source of rhythmicity and locomotor activity. Brief firing of PDF cells at different times of day generates a phase response curve (PRC), which mimics a light-mediated PRC and requires PDF receptor expression in the 5 E neurons. Firing also resembles light by causing Tim degradation in downstream neurons. Unlike light however, firing-mediated phase-shifting is Cry-independent and exploits the E3 ligase component Cul-3 in the early night to degrade Tim. These results suggest that PDF neurons integrate light information and then modulate the phase of E cell oscillations and behavioral rhythms. The results also explain how fly brain rhythms persist in constant darkness and without Cry.

Blaquiere, J. A., Lee, W. and Verheyen, E. M. (2014). Hipk promotes photoreceptor differentiation through the repression of Twin of eyeless and Eyeless expression. Dev Biol 390: 14-25. PubMed ID: 24631217
Summary: Organogenesis is a complex developmental process, which requires tight regulation of selector gene expression to specify individual organ types. The Pax6 homolog Eyeless (Ey) is an example of such a factor and its expression pattern reveals it is dynamically controlled during development. Eys paralog Twin of eyeless (Toy) induces its expression during embryogenesis, and the two genes are expressed in nearly identical patterns during the larval stages of development. While Ey must be expressed to initiate retinal specification, it must subsequently be repressed behind the morphogenetic furrow to allow for neuronal differentiation. Thus far, a few factors have been implicated in this repression including the signaling pathways Hedgehog (Hh) and Decapentaplegic (Dpp), and more recently downstream components of the retinal determination gene network (RDGN) Sine oculis (So), Eyes absent (Eya), and Dachshund (Dac). Homeodomain-interacting protein kinase (Hipk), a conserved serine-threonine kinase, regulates numerous factors during tissue patterning and development, including the Hh pathway. Using genetic analyses Hipk was identified a repressor of both Toy and Ey, and it may do so, in part, through Hh signaling. Evidence is provided that Ey repression is a critical step in ectopic eye development and that Hipk plays an important role in this process. Because Ey repression within the retinal field is a critical step in eye development, it is proposed that Hipk is a key link between eye specification and patterning.

Danielsen, E. T., Moeller, M. E., Dorry, E., Komura-Kawa, T., Fujimoto, Y., Troelsen, J. T., Herder, R., O'Connor, M. B., Niwa, R. and Rewitz, K. F. (2014). Transcriptional control of steroid biosynthesis genes in the Drosophila prothoracic gland by ventral veins lacking and knirps. PLoS Genet 10: e1004343. PubMed ID: 24945799
Summary: Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. This study shows that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. This study also showed that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. The data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

DeMill, C. M., Qiu, X., Kisiel, M., Bolotta, A. and Stewart, B. A. (2014). Investigation of the juxtamembrane region of neuronal-Synaptobrevin in synaptic transmission at the Drosophila neuromuscular junction. J Neurophysiol [Epub ahead of print]. PubMed ID: 24944220
Summary: The juxtamembrane region of the Drosophila SNARE protein, neuronal-Synaptobrevin (n-Syb) was tested for its role in synaptic transmission. A transgenic approach was used to express n-Syb mutant genes. The transgenes carried engineered point mutations that alter the amino acid sequence of the conserved tryptophan residues in the juxtamembrane sequence. Such transgenes were expressed in an n-syb hypomorphic background, which produce little endogenous protein. On their own, hypomorphic flies displayed severe motor inhibition, limited lifespan, reduced evoked junctional potentials (EJPs), decreased synchronicity in EJP time to peak and potentiation of EJPs with 10Hz stimulation. All of these deficits were restored to wild-type levels with the expression of wild-type transgenic n-syb, regulated by the endogenous promoter (n-sybWT). Transgenic mutants were created with one additional tryptophan (n-sybWW) or one less tryptophan (n-sybAA) than the wild-type sequence. While n-sybWW resembled n-sybWT in all variables listed, n-sybAA exhibited decreased EJP amplitude, synchronicity and quantal content. To determine whether the n-syb juxtamembrane region is important for transduction of force arising from SNARE complex assembly during membrane fusion a short 6 amino acid (n-sybL6) or long 24 amino acid (n-sybL24) flexible linkers were introduced into the n-syb transgene. A reduced EJP amplitude was observed in n-sybL6 but not n-sybL24, while both linker mutants showed a decreased quantal content and an effect on the readily releasable and recycling vesicle pools. In conclusion, mutation of the juxtamembrane region of n-sybdeleteriously affected synaptic transmission at the Drosophila neuromuscular junction.

Tuesday, July 24rd

Garaulet, D. L., Castellanos, M. C., Bejarano, F., Sanfilippo, P., Tyler, D. M., Allan, D. W., Sanchez-Herrero, E. and Lai, E. C. (2014). Homeotic Function of Drosophila Bithorax-Complex miRNAs Mediates Fertility by Restricting Multiple Hox Genes and TALE Cofactors in the CNS. Dev Cell. PubMed ID: 24909902
Summary: The Drosophila Bithorax complex (BX-C) Hox cluster contains a bidirectionally transcribed miRNA locus, and a deletion mutant (Δmir) lays no eggs and is completely sterile. This study shows that these miRNAs are expressed and active in distinct spatial registers along the anterior-posterior axis in the CNS. Δmir larvae derepress a network of direct homeobox gene targets in the posterior ventral nerve cord (VNC), including BX-C genes and their TALE cofactors, Homothorax and Extradenticle. These are phenotypically critical targets, because sterility of Δmir mutants was substantially rescued by heterozygosity of these genes. The posterior VNC contains Ilp7+ oviduct motoneurons, whose innervation and morphology are defective in Δmir females, are substantially rescued by heterozygosity of Δmir targets, especially within the BX-C. Collectively, this study reveals (1) critical roles for Hox miRNAs that determine segment-specific expression of homeotic genes, which are not masked by transcriptional regulation; and (2) that BX-C miRNAs are essential for neural patterning and reproductive behavior.

Fan, Y., Gittis, A. H., Juge, F., Qiu, C., Xu, Y. Z. and Rabinow, L. (2014). Multifunctional RNA Processing Protein SRm160 Induces Apoptosis and Regulates Eye and Genital Development in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 24907259
Summary: SRm160 is an SR-like protein implicated in multiple steps of RNA processing and nucleocytoplasmic export. Although its biochemical functions have been extensively described, its genetic interactions and potential participation in signaling pathways remain largely unknown despite the fact that it is highly phosphorylated in both mammalian cells and Drosophila. To begin elucidating the functions of the protein in signaling and its potential role in developmental processes, this study characterized mutant and over-expression SRm160 phenotypes in Drosophila and their interactions with the locus encoding the LAMMER protein kinase, Doa. SRm160 mutations are recessive lethal, while its over-expression generates phenotypes including roughened eyes and highly disorganized internal eye structure, which are due at least in part to aberrantly high levels of apoptosis. SRm160 is required for normal somatic sex determination, since its alleles strongly enhance a subtle sex transformation phenotype induced by Doa kinase alleles. Moreover, modification of SRm160 by DOA kinase appears to be necessary for its activity, since Doa alleles suppress phenotypes induced by SRm160 over-expression in the eye and enhance those in genital discs. Modification of SRm160 may occur through direct interaction because DOA kinase phosphorylates it in vitro. Remarkably, SRm160 protein was concentrated in the nuclei of pre-cellular embryos but was very rapidly excluded from nuclei or degraded coincident with cellularization. Also of interest, transcripts are restricted almost exclusively to the developing nervous system in mature embryos.

Atikukke, G., Albosta, P., Zhang, H. and Finley, R. L., Jr. (2014). A role for Drosophila Cyclin J in oogenesis revealed by genetic interactions with the piRNA pathway. Mech Dev [Epub ahead of print]. PubMed ID: 24946235
Summary: Cyclin J (CycJ) is a poorly characterized member of the Cyclin superfamily of cyclin-dependent kinase regulators, many of which regulate the cell cycle or transcription. Although CycJ is conserved in metazoans its cellular function has not been identified and no mutant defects have been described. In Drosophila, CycJ transcript is present primarily in ovaries and very early embryos, suggesting a role in one or both of these tissues. The CycJ gene (CycJ) lies immediately downstream of armitage (armi), a gene involved in the Piwi-associated RNA (piRNA) pathways that are required for silencing transposons in the germline and adjacent somatic cells. Mutations in armi result in oogenesis defects but a role for CycJ in oogenesis has not been defined. This study assessed oogenesis in CycJ mutants in the presence or absence of mutations in armi or other piRNA pathway genes. CycJ null ovaries appeared normal, indicating that CycJ is not essential for oogenesis under normal conditions. In contrast, armi null ovaries produced only two egg chambers per ovariole and the eggs had severe axis specification defects, as observed previously for armi and other piRNA pathway mutants. Surprisingly, the CycJ;armi double mutant failed to produce any mature eggs. The double null ovaries generally had only one egg chamber per ovariole and the egg chambers frequently contained an overabundance of differentiated germline cells. Production of these compound egg chambers could be suppressed with CycJ transgenes but not with mutations in the checkpoint gene mnk/loki, which suppress oogenesis defects in armi mutants. The CycJ null showed similar genetic interactions with the germline and somatic piRNA pathway gene piwi, and to a lesser extent with aubergine (aub), a member of the germline-specific piRNA pathway. The strong genetic interactions between CycJ and piRNA pathway genes reveal a role for CycJ in early oogenesis. These results suggest that CycJ is required to regulate egg chamber production or maturation when piRNA pathways are compromised.

Mohammed, J., Bortolamiol-Becet, D., Flynt, A. S., Gronau, I., Siepel, A. and Lai, E. C. (2014). Adaptive evolution of testis-specific, recently evolved, clustered miRNAs in Drosophila. RNA [Epub ahead of print]. PubMed ID: 24942624
Summary: The propensity of animal miRNAs to regulate targets bearing modest complementarity, most notably via pairing with miRNA positions approximately 2-8 (the 'seed'), is believed to drive major aspects of miRNA evolution. First, minimal targeting requirements have allowed most conserved miRNAs to acquire large target cohorts, thus imposing strong selection on miRNAs to maintain their seed sequences. Second, the modest pairing needed for repression suggests that evolutionarily nascent miRNAs may generally induce net detrimental, rather than beneficial, regulatory effects. Hence, levels and activities of newly emerged miRNAs are expected to be limited to preserve the status quo of gene expression. This study unexpectedly showed that Drosophila testes specifically express a substantial miRNA population that contravenes these tenets. Multiple genomic clusters of testis-restricted miRNAs harbor recently evolved miRNAs, whose experimentally verified orthologs exhibit divergent sequences, even within seed regions. Moreover, this class of miRNAs exhibits higher expression and greater phenotypic capacities in transgenic misexpression assays than do non-testis-restricted miRNAs of similar evolutionary age. These observations suggest that these testis-restricted miRNAs may be evolving adaptively, and several methods of evolutionary analysis provide strong support for this notion. Consistent with this, proof-of-principle tests show that orthologous miRNAs with divergent seeds can distinguish target sensors in a species-cognate manner. Finally, it was observed that testis-restricted miRNA clusters exhibit extraordinary dynamics of miRNA gene flux in other Drosophila species. Altogether, these findings reveal a surprising tissue-directed influence of miRNA evolution, involving a distinct mode of miRNA function connected to adaptive gene regulation in the testis.

Monday, June 23th

Li, L., Tian, X., Zhu, M., Bulgari, D., Bohme, M. A., Goettfert, F., Wichmann, C., Sigrist, S. J., Levitan, E. S. and Wu, C. (2014). Drosophila Syd-1, Liprin-α, and Protein Phosphatase 2A B' Subunit Wrd Function in a Linear Pathway to Prevent Ectopic Accumulation of Synaptic Materials in Distal Axons. J Neurosci 34: 8474-8487. PubMed ID: 24948803
Summary: During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-alpha in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, a proteomic screen was performed of Liprin-α-interacting proteins in Drosophila brains. Protein phosphatase 2A (PP2A; see Microtubule star) regulatory subunit B' [Wrd (Well Rounded)] was identified as a Liprin-α-interacting protein, and Wrd was demonstrated to mediate the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, data is provided suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot).

Brace, E. J., Wu, C., Valakh, V. and DiAntonio, A. (2014). SkpA restrains synaptic terminal growth during development and promotes axonal degeneration following injury. J Neurosci 34: 8398-8410. PubMed ID: 24948796
Summary: The Wallenda (Wnd)/dual leucine zipper kinase (DLK)-Jnk pathway is an evolutionarily conserved MAPK signaling pathway that functions during neuronal development and following axonal injury. Improper pathway activation causes defects in axonal guidance and synaptic growth, whereas loss-of-function mutations in pathway components impairs axonal regeneration and degeneration after injury. Regulation of this pathway is in part through the E3 ubiquitin ligase Highwire (Hiw), which targets Wnd/DLK for degradation to limit MAPK signaling. To explore mechanisms controlling Wnd/DLK signaling, a large-scale genetic screen was performed in Drosophila to identify negative regulators of the pathway. This study describes the identification and characterization of SkpA, a core component of SCF E3 ubiquitin ligases. Mutants in SkpA display synaptic overgrowth and an increase in Jnk signaling, similar to hiw mutants. The combination of hypomorphic alleles of SkpA and hiw leads to enhanced synaptic growth. Mutants in the Wnd-Jnk pathway suppress the overgrowth of SkpA mutants demonstrating that the synaptic overgrowth is due to increased Jnk signaling. These findings support the model that SkpA and the E3 ligase Hiw function as part of an SCF-like complex that attenuates Wnd/DLK signaling. In addition, SkpA, like Hiw, is required for synaptic and axonal responses to injury. Synapses in SkpA mutants are more stable following genetic or traumatic axonal injury, and axon loss is delayed in SkpA mutants after nerve crush. As in highwire mutants, this axonal protection requires Nmnat. Hence, SkpA is a novel negative regulator of the Wnd-Jnk pathway that functions with Hiw to regulate both synaptic development and axonal maintenance.

Winkle, C. C., McClain, L. M., Valtschanoff, J. G., Park, C. S., Maglione, C. and Gupton, S. L. (2014). A novel Netrin-1-sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching. J Cell Biol 205: 217-232. PubMed ID: 24778312
Summary: Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 (see Drosophila Netrins) promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. This study demonstrates that SNARE-mediated exocytosis is a prerequisite for axon branching and identifies the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. A direct interaction was identified between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1-sensitive interaction between TRIM9 and the SNARE component SNAP25 (see Drosophila Snap25). The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. These data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

Vogt, D., Hunt, R. F., Mandal, S., Sandberg, M., Silberberg, S. N., Nagasawa, T., Yang, Z., Baraban, S. C. and Rubenstein, J. L. (2014). Lhx6 directly regulates Arx and CXCR7 to determine cortical interneuron fate and laminar position. Neuron 82: 350-364. PubMed ID: 24742460
Summary: Cortical GABAergic interneurons have essential roles for information processing and their dysfunction is implicated in neuropsychiatric disorders. Transcriptional codes are elucidating mechanisms of interneuron specification in the MGE (a subcortical progenitor zone), which regulate their migration, integration, and function within cortical circuitry. Lhx6, a LIM-homeodomain transcription factor, is essential for specification of MGE-derived somatostatin and parvalbumin interneurons. This study demonstrates that some Lhx6minus MGE cells acquire a CGE-like fate. Using an in vivo MGE complementation/transplantation assay, this study shows that Lhx6-regulated genes Arx (Drosophila homolog: Aristaless) and atypical chemokine receptor CXCR7 rescue divergent aspects of Lhx6minus cell-fate and laminar mutant phenotypes and provide insight into a neonatal role for CXCR7 in MGE-derived interneuron lamination. Finally, Lhx6 directly binds in vivo to an Arx enhancer and to an intronic CXCR7 enhancer that remains active in mature interneurons. These data define the molecular identity of Lhx6 mutants and introduce technologies to test mechanisms in GABAergic interneuron differentiation.

Sunday, June 22nd

Weber, U., Rodriguez, E., Martignetti, J. and Mlodzik, M. (2014). Luna, a Drosophila KLF6/KLF7, Is Maternally Required for Synchronized Nuclear and Centrosome Cycles in the Preblastoderm Embryo. PLoS One 9: e96933. PubMed ID: 24915236
Summary: Kruppel like factors (KLFs) are conserved transcription factors that have been implicated in many developmental processes including differentiation, organ patterning, or regulation of stem cell pluripotency. This paper reports the generation and analysis of loss-of-function mutants of Drosophila Klf6/7, the luna gene. luna mutants are associated with very early embryonic defects prior to cellularization at the syncytial stage and cause DNA separation defects during the rapid mitotic cycles resulting in un-coupled DNA and centrosome cycles. These defects manifest themselves, both in animals that are maternally homozygous and heterozygous mutant. Surprisingly, luna is only required during the syncytial stages and not later in development, suggesting that the DNA segregation defect is linked to centrosomes, since centrosomes are dispensable for later cell divisions.

Foo, S. M., Sun, Y., Lim, B., Ziukaite, R., O'Brien, K., Nien, C. Y., Kirov, N., Shvartsman, S. Y. and Rushlow, C. A. (2014). Zelda Potentiates Morphogen Activity by Increasing Chromatin Accessibility. Curr Biol [Epub ahead of print]. PubMed ID: 24909324
Summary: Zygotic genome activation (ZGA) is a major genome programming event whereby the cells of the embryo begin to adopt specified fates. Experiments in Drosophila and zebrafish have revealed that ZGA depends on transcription factors that provide large-scale control of gene expression by direct and specific binding to gene regulatory sequences. Zelda (Zld) plays such a role in the Drosophila embryo, where it has been shown to control the action of patterning signals; however, the mechanisms underlying this effect remain largely unclear. A recent model proposed that Zld binding sites act as quantitative regulators of the spatiotemporal expression of genes activated by Dorsal (Dl), the morphogen that patterns the dorsoventral axis. This model was tested experimentally, using enhancers of brinker (brk) and short gastrulation (sog), both of which are directly activated by Dl, but at different concentration thresholds. In agreement with the model, it was shown that there is a clear positive correlation between the number of Zld binding sites and the spatial domain of enhancer activity. Likewise, the timing of expression could be advanced or delayed. Evidence is presented that Zld facilitates binding of Dl to regulatory DNA, and that this is associated with increased chromatin accessibility. Importantly, the change in chromatin accessibility is strongly correlated with the change in Zld binding, but not Dl. It is proposed that the ability of genome activators to facilitate readout of transcriptional input is key to widespread transcriptional induction during ZGA.

Wong, M. C., Dobi, K. C. and Baylies, M. K. (2014). Discrete Levels of Twist Activity Are Required to Direct Distinct Cell Functions during Gastrulation and Somatic Myogenesis. PLoS One 9: e99553. PubMed ID: 24915423
Summary: Twist (Twi), a conserved basic helix-loop-helix transcriptional regulator, directs the epithelial-to-mesenchymal transition (EMT), and regulates changes in cell fate, cell polarity, cell division and cell migration in organisms from flies to humans. Analogous to its role in EMT, Twist has been implicated in metastasis in numerous cancer types, including breast, pancreatic and prostate. In the Drosophila embryo, Twist is essential for discrete events in gastrulation and mesodermal patterning. This study derived a twi allelic series by examining the various cellular events required for gastrulation in Drosophila. By genetically manipulating the levels of Twi activity during gastrulation, it was found that coordination of cell division is the most sensitive cellular event, whereas changes in cell shape are the least sensitive. Strikingly, it was shown that by increasing levels of Snail expression in a severe twi hypomorphic allelic background, but not a twi null background, gastrulation can be reconstituted and viable adult flies produced. These results demonstrate that the level of Twi activity determines whether the cellular events of ventral furrow formation, EMT, cell division and mesodermal migration occur.

Swope, D., Kramer, J., King, T. R., Cheng, Y. S. and Kramer, S. G. (2014). Cdc42 is required in a genetically distinct subset of cardiac cells during drosophila dorsal vessel closure. Dev Biol [Epub ahead of print]. PubMed ID: 24949939
Summary: The embryonic heart tube is formed by the migration and subsequent midline convergence of two bilateral heart fields. In Drosophila the heart fields are organized into two rows of cardioblasts (CBs). While morphogenesis of the dorsal ectoderm, which lies directly above the Drosophila dorsal vessel (DV), has been extensively characterized, the migration and concomitant fundamental factors facilitating DV formation remain poorly understood. This study provides evidence that DV closure occurs at multiple independent points along the A-P axis of the embryo in a 'buttoning' pattern, divergent from the zippering mechanism observed in the overlying epidermis during dorsal closure. Moreover, it was demonstrated that a genetically distinct subset of CBs is programmed to make initial contact with the opposing row. To elucidate the cellular mechanisms underlying this process, the role of Rho GTPases was examined during cardiac migration using inhibitory and overexpression approaches. It was found that Cdc42 shows striking cell-type specificity during DV formation. Disruption of Cdc42 function specifically prevents CBs that express the homeobox gene tinman from completing their dorsal migration, resulting in a failure to make connections with their partnering CBs. Conversely, neighboring CBs that express the orphan nuclear receptor, seven-up, are not sensitive to Cdc42 inhibition. Furthermore, this phenotype was specific to Cdc42 and was not observed upon perturbation of Rac or Rho function. Together with the observation that DV closure occurs through the initial contralateral pairing of tinman-expressing CBs, these studies suggest that the distinct buttoning mechanism proposed for DV closure is elaborated through signaling pathways regulating Cdc42 activity in this cell type.

Saturday, June 21st

Schilling, S., Steiner, S., Zimmerli, D. and Basler, K. (2014). A regulatory receptor network directs the range and output of the Wingless signal. Development 141: 2483-2493. PubMed ID: 24917503
Summary: The potent activity of Wnt/Wingless (Wg) signals necessitates sophisticated mechanisms that spatially and temporally regulate their distribution and range of action. The two main receptor components for Wg - Arrow (Arr) and Frizzled 2 (Fz2) - are transcriptionally downregulated by Wg signaling, thus forming gradients that oppose that of Wg. This study analyze the relevance of this transcriptional regulation for the formation of the Wg gradient in the Drosophila wing disc by combining in vivo receptor overexpression with an in silico model of Wg receptor interactions. The experiments show that ubiquitous upregulation of Arr and Fz2 has no significant effects on Wg output, whereas clonal overexpression of these receptors leads to signaling discontinuities that have detrimental phenotypic consequences. These findings are supported by an in silico model for Wg diffusion and signal transduction, which suggests that abrupt changes in receptor levels causes discontinuities in Wg signaling. Furthermore, a 200 bp regulatory element in the arr locus was identified that can account for the Arr gradient, and it was shown that this is indirectly negatively controlled by Wg activity. Finally, the role of Frizzled 3 (Fz3) in was analyzed this system, and its expression, which is induced by Wg, was found to contribute to the establishment of the Arr and Fz2 gradients through counteracting canonical signaling. Taken together, these results provide a model in which the regulatory network of Wg and the three receptor components account for the range and shape of this prototypical morphogen system.

Wang, X., Gupta, P., Fairbanks, J. and Hansen, D. (2014). Protein kinase CK2 both promotes robust proliferation and inhibits the proliferative fate in the C. elegans germ line. Dev Biol [Epub ahead of print]. PubMed ID: 24824786
Summary: Stem cells are capable of both self-renewal (proliferation) and differentiation. Determining the regulatory mechanisms controlling the balance between stem cell proliferation and differentiation is not only an important biological question, but also holds the key for using stem cells as therapeutic agents. The Caenorhabditis elegans germ line has emerged as a valuable model to study the molecular mechanisms controlling stem cell behavior. This study describes a large-scale RNAi screen that identified kin-10, which encodes the beta subunit of protein kinase CK2 (see Drosophila CK2), as a novel factor regulating stem cell proliferation in the C. elegans germ line. While a loss of kin-10 in an otherwise wild-type background results in a decrease in the number of proliferative cells, loss of kin-10 in sensitized genetic backgrounds results in a germline tumor. Therefore, kin-10 is not only necessary for robust proliferation, it also inhibits the proliferative fate. A role or kin-10's regulatory role in inhibiting the proliferative fate is carried out through the CK2 holoenzyme, rather than through a holoenzyme-independent function, and that it functions downstream of GLP-1/Notch signaling. It is proposed that a loss of kin-10 leads to a defect in CK2 phosphorylation of its downstream targets, resulting in abnormal activity of target protein(s) that are involved in the proliferative fate vs. differentiation decision. This eventually causes a shift towards the proliferative fate in the stem cell fate decision.

Benkemoun, L., Descoteaux, C., Chartier, N. T., Pintard, L. and Labbe, J. C. (2014). PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo. J Cell Biol 205: 447-455. PubMed ID: 24841566
Summary: Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, this study found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 (See Drosophila Lkb1) and PAR-1 (see Drosophila Par1) polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. Our results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division.

Zhang, Y., Ding, Y., Chen, Y. G. and Tao, Q. (2014). NEDD4L regulates convergent extension movements in Xenopus embryos via Disheveled-mediated non-canonical Wnt signaling. Dev Biol 392(1):15-25. PubMed ID: 24833518
Summary: During the early vertebrate body plan formation, convergent extension (CE) of dorsal mesoderm and neurectoderm is coordinated by the evolutionarily conserved non-canonical Wnt/PCP signaling. Disheveled (Dvl; see Drosophila Disheveled), a key mediator of Wnt/PCP signaling, is essential for the medial-lateral polarity formation in the cells undergoing convergent extension movements. NEDD4L, a highly conserved HECT type E3 ligase, has been reported to regulate the stability of multiple substrates including Dvl2. This study demonstrates that NEDD4L is required for the cellular polarity formation and convergent extension in the early Xenopus embryos. Depletion of NEDD4L in early Xenopus embryos results in the loss of mediolateral polarity of the convergent-extending mesoderm cells and the shortened body axis, resembling those defects caused by the disruption of non-canonical Wnt signaling. Depletion of xNEDD4L also blocks the elongation of the animal explants in response to endogenous mesoderm inducing signals and partially compromises the expression of Brachyury. Importantly, reducing Dvl2 expression can largely rescue the cellular polarity and convergent extension defects in NEDD4L-depleted embryos and explants. Together with the data that NEDD4L reduces Dvl2 protein expression in the frog embryos, these findings suggest that regulation of Dvl protein levels by NEDD4L is essential for convergent extension during early Xenopus embryogenesis.

Friday, June 20th

Jeibmann, A., Eikmeier, K., Linge, A., Kool, M., Koos, B., Schulz, J., Albrecht, S., Bartelheim, K., Fruhwald, M. C., Pfister, S. M., Paulus, W. and Hasselblatt, M. (2014). Identification of genes involved in the biology of atypical teratoid/rhabdoid tumours using Drosophila melanogaster. Nat Commun 5: 4005. PubMed ID: 24892285
Summary: Atypical teratoid/rhabdoid tumours (AT/RT) are malignant brain tumours. Unlike most other human brain tumours, AT/RT are characterized by inactivation of one single gene, SMARCB1. SMARCB1 is a member of the evolutionarily conserved SWI/SNF chromatin remodelling complex, which has an important role in the control of cell differentiation and proliferation. Little is known, however, about the pathways involved in the oncogenic effects of SMARCB1 inactivation, which might also represent targets for treatment. This study reports a comprehensive genetic screen in the fruit fly that revealed several genes not yet associated with loss of snr1, the Drosophila homologue of SMARCB1. This study confirmed the functional role of identified genes (including merlin, kibra and expanded, known to regulate hippo signalling pathway activity) in human rhabdoid tumour cell lines and AT/RT tumour samples. These results demonstrate that fly models can be employed for the identification of clinically relevant pathways in human cancer.

Chen, A. Y., Xia, S., Wilburn, P. and Tully, T. (2014). Olfactory deficits in an alpha-synuclein fly model of Parkinson's disease. PLoS One 9: e97758. PubMed ID: 24879013
Summary: Parkinson's disease (PD) is the most common motor neurodegenerative disorder. Olfactory dysfunction is a prevalent feature of PD. It often precedes motor symptoms by several years and is used in assisting PD diagnosis. However, the cellular and molecular bases of olfactory dysfunction in PD are not known. The fruit fly expressing human alpha-synuclein protein or its mutant, A30P, captures several hallmarks of PD and has been successfully used to model PD in numerous studies. First, olfactory deficits are reported in flys expressing A30P (A30P), showing deficits in two out of three olfactory modalities, tested - olfactory acuity and odor discrimination. The remaining third modality is odor identification/naming. Second, oxidative stress is an important environmental risk factor of PD. Oxidative stress was shown to exacerbated the two affected olfactory modalities in younger A30P flies. Third, different olfactory receptor neurons are activated differentially by different odors in flies. In a separate experiment, it was shown that the odor discrimination deficit in A30P flies is general and not restricted to a specific class of chemical structure. Lastly, by restricting A30P expression to dopamine, serotonin or olfactory receptor neurons, it was shown that A30P expression in dopamine neurons is necessary for development of both acuity and discrimination deficits, while serotonin and olfactory receptor neurons appeared not involved. These data demonstrate olfactory deficits in a synuclein fly PD model for exploring olfactory pathology and physiology, and for monitoring PD progression and treatment.

He, F., Krans, A., Freibaum, B. D., Taylor, J. P. and Todd, P. K. (2014). TDP-43 suppresses CGG repeat-induced neurotoxicity through interactions with HnRNP A2/B1. Hum Mol Genet [Epub ahead of print]. PubMed ID: 24920338
Summary: Nucleotide repeat expansions can elicit neurodegeneration as RNA by sequestering specific RNA-binding proteins, preventing them from performing their normal functions. Conversely, mutations in RNA-binding proteins can trigger neurodegeneration at least partly by altering RNA metabolism. In Fragile X-associated tremor/ataxia syndrome (FXTAS), a CGG repeat expansion in the 5'UTR of the fragile X gene (FMR1) leads to progressive neurodegeneration in patients and CGG repeats in isolation elicit toxicity in Drosophila and other animal models. This study has identified the amyotrophic lateral sclerosis (ALS)-associated RNA-binding protein TAR DNA-binding protein-43 homolog (TDP-43) as a suppressor of CGG repeat-induced toxicity in a Drosophila model of FXTAS. The rescue appears specific to TDP-43, as co-expression of another ALS-associated RNA-binding protein, FUS, exacerbates the toxic effects of CGG repeats. Suppression of CGG RNA toxicity was abrogated by disease-associated mutations in TDP-43. TDP-43 does not co-localize with CGG RNA foci and its ability to bind RNA is not required for rescue. TDP-43-dependent rescue does, however, require fly hnRNP A2/B1 homologues Hrb87F and Hrb98DE. Deletions in the C-terminal domain of TDP-43 that preclude interactions with hnRNP A2/B1 abolish TDP-43-dependent rescue of CGG repeat toxicity. In contrast, suppression of CGG repeat toxicity by hnRNP A2/B1 is not affected by RNAi-mediated knockdown of the fly TDP-43 orthologue, TBPH. Lastly, TDP-43 suppresses CGG repeat-triggered mis-splicing of an hnRNP A2/B1-targeted transcript. These data support a model in which TDP-43 suppresses CGG-mediated toxicity through interactions with hnRNP A2/B1 and suggest a convergence of pathogenic cascades between repeat expansion disorders and RNA-binding proteins implicated in neurodegenerative disease.

Linhart, R., Wong, S. A., Cao, J., Tran, M., Huynh, A., Ardrey, C., Park, J. M., Hsu, C., Taha, S., Peterson, R., Shea, S., Kurian, J. and Venderova, K. (2014). Vacuolar protein sorting 35 (Vps35) rescues locomotor deficits and shortened lifespan in Drosophila expressing a Parkinson's disease mutant of Leucine-rich repeat kinase 2 (LRRK2). Mol Neurodegener 9: 23. PubMed ID: 24915984
Summary: Parkinson's disease (PD) is the most common movement neurodegenerative movement disorder. An incomplete understanding of the molecular pathways involved in its pathogenesis impedes the development of effective disease-modifying treatments. To address this gap, a Drosophila model of PD has been generated that overexpresses PD pathogenic mutant form of the second most common causative gene of PD, Leucine-Rich Repeat Kinase 2 (LRRK2). This model was employed in a genetic modifier screen and identified a gene that encodes for a core subunit of retromer - a complex essential for the sorting and recycling of specific cargo proteins from endosomes to the trans-Golgi network and cell surface. Evidence is presented that overexpression of the Vps35 or Vps26 component of the cargo-recognition subunit of the retromer complex ameliorates the pathogenic mutant LRRK2 eye phenotype. Furthermore, overexpression of Vps35 or Vps26 significantly protects from the locomotor deficits observed in mutant LRRK2 flies, as assessed by the negative geotaxis assay, and rescues their shortened lifespan. Strikingly, overexpressing Vps35 alone protects from toxicity of rotenone, a neurotoxin commonly used to model parkinsonism, both in terms of lifespan and locomotor activity of the flies, and this protection is sustained and even augmented in the presence of mutant LRRK2. Finally, it was demonstrated that knocking down expression of Vps35 in dopaminergic neurons causes a significant locomotor impairment. From these results it is concluded that LRRK2 plays a role in the retromer pathway and that this pathway is involved in PD pathogenesis.

Thursday, June 19th

Zhang, Z., Wang, J., Schultz, N., Zhang, F., Parhad, S. S., Tu, S., Vreven, T., Zamore, P. D., Weng, Z. and Theurkauf, W. E. (2014). The HP1 Homolog Rhino Anchors a Nuclear Complex that Suppresses piRNA Precursor Splicing. Cell 157: 1353-1363. PubMed ID: 24906152
Summary: piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. Rhino is shown to bind specifically to the heterochromatic clusters that produce piRNA precursors; binding directly correlates with piRNA production. Rhino colocalizes to germline nuclear foci with Rai1/DXO-related protein Cutoff (Cuff) and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced and that rhino, cuff, and uap56 mutations increase expression of spliced cluster transcripts over 100-fold. LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. It is therefore proposed that Rhino anchors a nuclear complex that suppresses cluster transcript splicing and it is speculated that stalled splicing differentiates piRNA precursors from mRNAs.

Mohn, F., Sienski, G., Handler, D. and Brennecke, J. (2014). The Rhino-Deadlock-Cutoff Complex Licenses Noncanonical Transcription of Dual-Strand piRNA Clusters in Drosophila. Cell 157: 1364-1379. PubMed ID: 24906153
Summary: Argonaute proteins of the PIWI clade (see Drosophila Piwi) are central to transposon silencing in animal gonads. Their target specificity is defined by 23-30 nt PIWI interacting RNAs (piRNAs), which mostly originate from discrete genomic loci termed piRNA clusters. This study shows that a complex composed of Rhino, Deadlock, and Cutoff (RDC) defines dual-strand piRNA clusters genome-wide in Drosophila ovaries. The RDC is anchored to H3K9me3-marked chromatin in part via Rhino's chromodomain. Depletion of Piwi results in loss of the RDC and small RNAs at a subset of piRNA clusters, demonstrating a feedback loop between Piwi and piRNA source loci. Intriguingly, profiles of RNA polymerase II occupancy, nascent transcription, and steady-state RNA levels reveal that the RDC licenses noncanonical transcription of dual-strand piRNA clusters. Likely, this process involves 5' end protection of nascent RNAs and suppression of transcription termination. These data provide key insight into the regulation and evolution of piRNA clusters.

Xiol, J., Spinelli, P., Laussmann, M. A., Homolka, D., Yang, Z., Cora, E., Coute, Y., Conn, S., Kadlec, J., Sachidanandam, R., Kaksonen, M., Cusack, S., Ephrussi, A. and Pillai, R. S. (2014). RNA Clamping by Vasa Assembles a piRNA Amplifier Complex on Transposon Transcripts. Cell [Epub ahead of print]. PubMed ID: 24910301
Summary: Germline-specific Piwi-interacting RNAs (piRNAs) protect animal genomes against transposons and are essential for fertility. piRNAs targeting active transposons are amplified by the ping-pong cycle, which couples Piwi endonucleolytic slicing of target RNAs to biogenesis of new piRNAs. This study describes the identification of a transient Amplifier complex that mediates biogenesis of secondary piRNAs in insect cells. Amplifier is nucleated by the DEAD box RNA helicase Vasa and contains the two Piwi proteins participating in the ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasa's helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. ATP-dependent RNP remodeling by Vasa facilitates transfer of 5' sliced piRNA precursors between ping-pong partners, and loss of this activity causes sterility in Drosophila. These results reveal the molecular basis for the small RNA amplification that confers adaptive immunity against transposons.

Van Wynsberghe, P. M., Finnegan, E. F., Stark, T., Angelus, E. P., Homan, K. E., Yeo, G. W. and Pasquinelli, A. E. (2014). The Period protein homolog LIN-42 negatively regulates microRNA biogenesis in C. elegans. Dev Biol 390: 126-135. PubMed ID: 24699545
Summary: MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate gene expression in many multicellular organisms. They are encoded in the genome and transcribed into primary (pri-) miRNAs before two processing steps that ultimately produce the mature miRNA. In order to generate the appropriate amount of a particular miRNA in the correct location at the correct time, proper regulation of miRNA biogenesis is essential. This study identifies the Period protein homolog LIN-42 as a new regulator of miRNA biogenesis in Caenorhabditis elegans. A spontaneous suppressor of the normally lethal let-7n2853 allele (see Drosophila let-7) was mapped to the lin-42 gene. Mutations in this allele (ap201) or a second lin-42 allele (n1089) caused increased mature let-7 miRNA levels at most time points when mature let-7 miRNA is normally expressed. Levels of pri-let-7 and a let-7 transcriptional reporter were also increased in lin-42n1089 worms. These results indicate that LIN-42 normally represses pri-let-7 transcription and thus the accumulation of let-7 miRNA. This inhibition is not specific to let-7, as pri- and mature levels of lin-4 and miR-35 were also increased in lin-42 mutants. Furthermore, small RNA-seq analysis showed widespread increases in the levels of mature miRNAs in lin-42 mutants. Thus, it is proposed that the period protein homolog LIN-42 is a global regulator of miRNA biogenesis.

Wednesday, June 18th

Glossop, N. R., Gummadova, J. O., Ghangrekar, I., Hardin, P. E. and Coutts, G. A. (2014). Effects of Twin-of-Eyeless on Clock Gene Expression and Central-Pacemaker Neuron Development in Drosophila. J Biol Rhythms 29: 151-166. PubMed ID: 24916389
Summary: Circadian oscillators are autonomous molecular rhythms that reside in cells to align whole-organism physiology and behavior to the 24-h day. In flies, as in mammals, the oscillator operates in cells that coexpress Clock (Clk) and Cycle (Cyc). Recent work in Drosophila has shown that Clk is unique in its ability to generate heterologous oscillators, indicating that Clk gene expression defines the circadian cell fate. Using standard in vitro and in vivo techniques this study shows that Twin-of-Eyeless (Toy) regulates Clk expression in small ventrolateral neurons (s-LNvs) that coordinate sleep-wake cycles. Crucially, toy binds multiple sites at the Clk locus, is expressed independent of Clk-Cyc in LNvs, regulates Clk protein levels under optimal photoperiodic conditions, and sets clock-speed during endogenous free-run. Furthermore, Toy is necessary for the onset of Clk expression in LNvs during embryogenesis. It is proposed that Toy contributes to a transcription complex that functions upstream of the oscillator to promote Clk expression in s-LNvs.

Kimura, S., Sakakibara, Y., Sato, K., Ote, M., Ito, H., Koganezawa, M. and Yamamoto, D. (2014). The Drosophila Lingerer protein cooperates with Orb2 in long-term memory formation. J Neurogenet: 1-41. PubMed ID: 24913805
Summary: Recently mated Drosophila females were shown to be reluctant to copulate and to exhibit rejecting behavior when courted by a male. Males that experience mate refusal by a mated female subsequently attenuate their courtship effort toward not only mated females but also virgin females. This courtship suppression persists for more than a day, and thus represents long-term memory. The courtship long-term memory has been shown to be impaired in heterozygotes as well as homozygotes of mutants in orb2, a locus encoding a set of CPEB RNA-binding proteins. This study shows that the impaired courtship long-term memory in orb2-mutant heterozygotes is restored by reducing the activity of lingerer (lig), another putative RNA-binding protein gene, yet on its own the loss-of-function lig mutation is without effect. It was further shown that Lig forms a complex with Orb2. It is inferred that a reduction in the Lig levels compensates the Orb2 deficiency by mitigating the negative feedback for Orb2 expression and thereby alleviating defects in long-term memory.

Sen, A., Grimm, S., Hofmeyer, K. and Pflugfelder, G. O. (2014). Optomotor-blind in the development of the Drosophila HS and VS lobula plate tangential cells. J Neurogenet: 1-49. PubMed ID: 24912380
Summary: The horizontal system and vertical system cells of the dipteran optic lobes are well understood regarding their physiology and role in visually guided behaviour. Little is known, however, about their development. Drosophila optomotor-blind (omb) is required for the development of the HS/VS (Horizontally Sensitive/Vertically Sensitive) cells which are lacking in the adult brain of the In(1)omb[H31] regulatory mutant. The omb regulatory region, required for HS/VS development, was analyzed for enhancers active in the central nervous system. A 1 kb fragment, ombJb, was identified 114 kb downstream of the omb transcription start site, that could drive expression in much of the presumptive embryonic optic lobe anlage. Expression in these cells is lost in In(1)omb[H31] suggesting that they contain the HS/VS precursor cell(s). Laser ablation was used in the embryonic CNS in order to localize the position of the HS/VS precursor cell(s) in this tissue. An omb-Gal4 enhancer trap line, which showed activity in the optic lobe anlage in a pattern similar to ombJb enhancer, was used to drive GFP expression, thus allowing focusing of the Laser beam to the relevant area. A small region in the embryonic brain was identified from which the HS/VS cells are likely to develop. Omb encodes a transcription factor of the T-box family. Since loss of omb disrupts HS/VS cell development, it is assumed that HS/VS ontogeny is controlled by Omb target genes. As a first step toward their identification, this study characterized the Omb DNA-binding specificity.

Schoofs, A., Huckesfeld, S., Surendran, S. and Pankratz, M. J. (2014). Serotonergic pathways in the Drosophila larval enteric nervous system. J Insect Physiol [Epub ahead of print]. PubMed ID: 24907674
Summary: The enteric nervous system is critical for coordinating diverse feeding-related behaviors and metabolism. This study has characterized a cluster of four serotonergic neurons in Drosophila larval brain: cell bodies are located in the subesophageal ganglion (SOG) whose neuronal processes project into the enteric nervous system. Electrophysiological, calcium imaging and behavioral analyses indicate a functional role of these neurons in modulating foregut motility. It is suggested that the axonal projections of this serotonergic cluster may be part of a brain-gut neural pathway that is functionally analogous to the vertebrate vagus nerve.

Tuesday, June 17th

Nguyen, M. M., McCracken, C. J., Milner, E. S., Goetschius, D. J., Weiner, A. T., Long, M. K., Michael, N. L., Munro, S. and Rolls, M. M. (2014). γ-tubulin controls neuronal microtubule polarity independently of Golgi outposts. Mol Biol Cell [Epub ahead of print]. PubMed ID: 24807906
Summary: Neurons have highly polarized arrangements of microtubules, but it is incompletely understood how microtubule polarity is controlled in either axons or dendrites. To explore whether microtubule nucleation by γ-tubulin might contribute to polarity, neuronal microtubules were examined in Drosophila containing gain- or loss-of-function alleles of γ-tubulin. Both increased and decreased activity of γ-tubulin, the core microtubule nucleation protein, altered microtubule polarity in axons and dendrites, suggesting a close link between regulation of nucleation and polarity. To test whether nucleation might locally regulate polarity in axons and dendrites, the distribution of γ-tubulin was examined. Consistent with local nucleation, tagged and endogenous γ-tubulin were found in specific positions in dendrites and axons. As the Golgi complex can house nucleation sites, whether microtubule nucleation might occur at dendritic Golgi outposts was explored. However, distinct Golgi outposts were not present in all dendrites that required regulated nucleation for polarity. Moreover, when the Golgi were dragged out of dendrites with an activated kinesin, γ-tubulin remained in dendrites. It is concluded that regulated microtubule nucleation controls neuronal microtubule polarity, but that the Golgi complex is not directly involved in housing nucleation sites.

Kitazawa, D., Matsuo, T., Kaizuka, K., Miyauchi, C., Hayashi, D. and Inoue, Y. H. (2014). Orbit/CLASP Is Required for Myosin Accumulation at the Cleavage Furrow in Drosophila Male Meiosis. PLoS One 9: e93669. PubMed ID: 24850412
Summary: Peripheral microtubules (MTs) near the cell cortex are essential for the positioning and continuous constriction of the contractile ring (CR) in cytokinesis. Time-lapse observations of Drosophila male meiosis showed that myosin II is first recruited along the cell cortex independent of MTs. Then, shortly after peripheral MTs make contact with the equatorial cortex, myosin II is concentrated there in a narrow band. After MT contact, anillin and F-actin abruptly appear on the equatorial cortex, simultaneously with myosin accumulation. The accumulation of myosin does not require centralspindlin, a protein complex that regulates several processes during cytokinesis, but is instead dependent on Orbit, a Drosophila ortholog of the MT plus-end tracking protein CLASP. This protein is required for stabilization of central spindle MTs, which are essential for cytokinesis. Orbit is also localized in a mid-zone of peripheral MTs, and is concentrated in a ring at the equatorial cortex during late anaphase. Fluorescence resonance energy transfer experiments indicated that Orbit is closely associated with F-actin in the CR. It was also shown that the myosin heavy chain is in close proximity with Orbit in the cleavage furrow region. Centralspindlin is dispensable in Orbit ring formation. Instead, the Polo-KLP3A/Feo complex is required for the Orbit accumulation independently of the Orbit MT-binding domain. However, orbit mutations of consensus sites for the phosphorylation of Cdk1 or Polo did not influence the Orbit accumulation, suggesting an indirect regulatory role of these protein kinases in Orbit localization. Orbit is also necessary for the maintenance of the CR. These data suggest that Orbit plays an essential role as a connector between MTs and the CR in Drosophila male meiosis.

Do, K. K., Hoang, K. L. and Endow, S. A. (2014). The kinesin-13 KLP10A motor regulates oocyte spindle length and affects EB1 binding without altering microtubule growth rates. Biol Open. PubMed ID: 24907370
Summary: Kinesin-13 motors are unusual in that they do not walk along microtubules, but instead diffuse to the ends, where they remove tubulin dimers, regulating microtubule dynamics. This study shows that Drosophila kinesin-13 klp10A regulates oocyte meiosis I spindle length and is haplo-insufficient - KLP10A, reduced by RNAi or a loss-of-function P element insertion mutant, results in elongated and mispositioned oocyte spindles, and abnormal cortical microtubule asters and aggregates. KLP10A knockdown by RNAi does not significantly affect microtubule growth rates in oocyte spindles, but, unexpectedly binding and unbinding of EB1, which controls the plus ends of growing microtubules, were slowed, suggesting a previously unobserved role for kinesin-13 in mediating EB1 binding interactions with microtubules. Kinesin-13 may regulate spindle length both by disassembling subunits from microtubule ends and facilitating EB1 binding to plus ends. An increased number of paused microtubules was observed in klp10A RNAi knockdown spindles, consistent with a reduced frequency of microtubule catastrophes. Overall, these findings indicate that reduced kinesin-13 decreases microtubule disassembly rates and affects EB1 interactions with microtubules, rather than altering microtubule growth rates, causing spindles to elongate and abnormal cortical microtubule asters and aggregates to form.

Bolkan, B. J. and Kretzschmar, D. (2014). Loss of Tau results in defects in photoreceptor development and progressive neuronal degeneration in Drosophila. Dev Neurobiol [Epub ahead of print]. PubMed ID: 24909306
Summary: Accumulations of Tau, a microtubule-associated protein, into neurofibrillary tangles is a hallmark of Alzheimer's disease and other tauopathies. However, the mechanisms leading to this pathology are still unclear: the aggregates themselves could be toxic or the sequestration of Tau into tangles might prevent Tau from fulfilling its normal functions, thereby inducing a loss of function defect. Surprisingly, the consequences of losing normal Tau expression in vivo are still not well understood, in part due to the fact that Tau knockout mice show only subtle phenotypes, presumably due to the fact that mammals express several MAPs with partially overlapping functions. In contrast, flies express fewer microtubule-associated proteins, with Tau being the only member of the Tau/MAP2/MAP4 family. Therefore, Drosophila was used to address the physiological consequences caused by the loss of Tau. Reducing the levels of fly Tau (dTau) ubiquitously resulted in developmental lethality, whereas deleting Tau specifically in neurons or the eye caused progressive neurodegeneration. Similarly, chromosomal mutations affecting dTau also caused progressive degeneration in both the eye and brain. Although photoreceptor cells initially developed normally in dTau knockdown animals, they subsequently degenerated during late pupal stages whereas weaker dTau alleles caused an age-dependent defect in rhabdomere structure. Expression of wild type human Tau partially rescued the neurodegenerative phenotype caused by the loss of endogenous dTau, suggesting that the functions of Tau proteins are functionally conserved from flies to humans.

Monday, June 16th

Simon, R., Aparicio, R., Housden, B. E., Bray, S. and Busturia, A. (2014). Drosophila p53 controls Notch expression and balances apoptosis and proliferation. Apoptosis [Epub ahead of print]. PubMed ID: 24858703
Summary: A balance between cell proliferation and apoptosis is important for normal development and tissue homeostasis. Under stress conditions, the conserved tumor suppressor and transcription factor Dp53 induces apoptosis to contribute to the maintenance of homeostasis. However, in some cases Dp53-induced apoptosis results in the proliferation of surrounding non-apoptotic cells. To gain insight into the Dp53 function in the control of apoptosis and proliferation, the interaction between the Drosophila Dp53 and Notch genes was studied. Evidence is presented that simultaneous reduction of Dp53 and Notch function synergistically increases the wing phenotype of Notch heterozygous mutant flies. Further, it was found that a Notch cis-regulatory element is responsive to loss and gain of Dp53 function and that over-expression of Dp53 up-regulates Notch mRNA and protein expression. These findings suggest not only that Dp53 and Notch act together to control wing development but also indicate that Dp53 transcriptionally regulates Notch expression. Moreover, using Notch gain and loss of function mutations the relevance of Dp53 and Notch interactions was studied in the process of Dp53-apoptosis induced proliferation. Results show that proliferation induced by Dp53 over-expression is dependent on Notch, thus identifying Notch as a new player in Dp53-induced proliferation. Interestingly, it was found that Dp53-induced Notch activation and proliferation occurs even under conditions where apoptosis is inhibited. These findings highlight the conservation between flies and vertebrates of the Dp53 and Notch cross-talk and suggest that Dp53 has a dual role regulating cell death and proliferation gene networks to control the homeostatic balance between apoptosis and proliferation.

DeVorkin, L., Go, N. E., Hou, Y. C., Moradian, A., Morin, G. B. and Gorski, S. M. (2014). The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB. J Cell Biol 205: 477-492. PubMed ID: 24862573
Summary: Increasing evidence reveals that a subset of proteins participates in both the autophagy and apoptosis pathways, and this intersection is important in normal physiological contexts and in pathological settings. This paper shows that the Drosophila effector caspase, Drosophila caspase 1 (Dcp-1), localizes within mitochondria and regulates mitochondrial morphology and autophagic flux. Loss of Dcp-1 leads to mitochondrial elongation, increased levels of the mitochondrial adenine nucleotide translocase Stress-sensitive B (SesB), increased adenosine triphosphate (ATP), and a reduction in autophagic flux. Moreover, it was found that SesB suppresses autophagic flux during midoogenesis, identifying a novel negative regulator of autophagy. Reduced SesB activity or depletion of ATP by oligomycin A could rescue the autophagic defect in Dcp-1 loss-of-function flies, demonstrating that Dcp-1 promotes autophagy by negatively regulating SesB and ATP levels. Furthermore, it was found that pro-Dcp-1 interacts with SesB in a nonproteolytic manner to regulate its stability. These data reveal a new mitochondrial-associated molecular link between nonapoptotic caspase function and autophagy regulation in vivo.

Kim, C. H., Paik, D., Rus, F. and Silverman, N. (2014). The caspase-8 homolog Dredd cleaves Imd and Relish but is not inhibited by p35. J Biol Chem [Epub ahead of print]. PubMed ID: 24891502
Summary: In Drosophila, the Imd pathway is activated by diaminopimelic acid (DAP)-type peptidoglycan and triggers the humoral innate immune response, including the robust induction of antimicrobial peptide gene expression. Imd and Relish, two essential components of this pathway, are both endoproteolytically cleaved upon immune stimulation. Genetic analyses have shown that these cleavage events are dependent on the caspase-8 like Dredd, suggesting that Imd and Relish are direct substrates of Dredd. Among the seven Drosophila caspases, this study found that Dredd uniquely promotes Imd and Relish processing, and purified recombinant Dredd cleaves Imd and Relish in vitro. In addition, interdomain cleavage of Dredd is not required for Imd or Relish processing and is not observed during immune stimulation. Baculovirus p35, a suicide substrate of executioner caspases, is not cleaved by purified Dredd in vitro. Consistent with this biochemistry but contrary to earlier reports, p35 does not interfere with Imd signaling in S2* cells or in vivo.

Chuang, C. L., Lu, Y. N., Wang, H. C. and Chang, H. Y. (2014). Genetic dissection reveals that Akt is the critical kinase downstream of LRRK2 to phosphorylate and inhibit FOXO1, and promotes neuron survival. Hum Mol Genet [Epub ahead of print]. PubMed ID: 24916379
Summary: Leucine-rich repeat kinase 2 (LRRK2) is a complex kinase and mutations in LRRK2 are perhaps the most common genetic cause of Parkinson's disease (PD). However, the identification of the normal physiological function of LRRK2 remains elusive. This study shows that LRRK2 protects neurons against apoptosis induced by the Drosophila genes grim, hid and reaper. Genetic dissection reveals that Akt is the critical downstream kinase of LRRK2 that phosphorylates and inhibits FOXO1, and thereby promotes survival. Like human LRRK2, Drosophila lrrk also promotes neuron survival; lrrk loss-of-function mutant displays reduced cell numbers, which can be rescued by LRRK2 expression. Importantly, LRRK2 G2019S and LRRK2 R1441C mutants impair the ability of LRRK2 to activate Akt, and result in a failure of preventing apoptotic death. Ectopic expression of a constitutive active form of Akt hence is sufficient to rescue this functional deficit. These data establish that LRRK2 can protect neurons from apoptotic insult through a survival pathway in which LRRK2 signals to activate Akt, and then inhibits FOXO1. These results might indicate that a therapeutic pathway to promote neuron survival and to prevent neurodegeneration in Parkinson's disease.

Sunday, June 15th

Luo, J., Lushchak, O. V., Goergen, P., Williams, M. J. and Nassel, D. R. (2014). Drosophila insulin-producing cells are differentially modulated by serotonin and octopamine receptors and affect social behavior. PLoS One 9: e99732. PubMed ID: 24923784
Summary: A set of 14 insulin-producing cells (IPCs) in the Drosophila brain produces three insulin-like peptides (DILP2, 3 and 5). Activity in IPCs and release of DILPs is nutrient dependent and controlled by multiple factors such as fat body-derived proteins, neurotransmitters, and neuropeptides. Two monoamine receptors, the octopamine receptor OAMB and the serotonin receptor 5-HT1A, are expressed by the IPCs. These receptors may act antagonistically on adenylate cyclase. This study investigated the action of the two receptors on activity in and output from the IPCs. Knockdown of OAMB by targeted RNAi led to elevated Dilp3 transcript levels in the brain, whereas 5-HT1A knockdown resulted in increases of Dilp2 and 5. OAMB-RNAi in IPCs leads to extended survival of starved flies and increased food intake, whereas 5-HT1A-RNAi produces the opposite phenotypes. However, knockdown of either OAMB or 5-HT1A in IPCs both lead to increased resistance to oxidative stress. In assays of carbohydrate levels it was found that 5-HT1A knockdown in IPCs resulted in elevated hemolymph glucose, body glycogen and body trehalose levels, while no effects were seen after OAMB knockdown. It was also found that manipulations of the two receptors in IPCs affected male aggressive behavior in different ways and 5-HT1A-RNAi reduced courtship latency. These observations suggest that activation of 5-HT1A and OAMB signaling in IPCs generates differential effects on Dilp transcription, fly physiology, metabolism and social interactions. However the findings do not support an antagonistic action of the two monoamines and their receptors in this particular system.

Pineiro, C., Lopes, C. S. and Casares, F. (2014). A conserved transcriptional network regulates lamina development in the Drosophila visual system. Development [Epub ahead of print]. PubMed ID: 24924198
Summary: The visual system of insects is a multilayered structure composed externally by the compound eye and internally by the three ganglia of the optic lobe: lamina, medulla and the lobula complex. The differentiation of lamina neurons depends heavily on Hedgehog (Hh) signaling, which is delivered by the incoming photoreceptor axons, and occurs in a wave-like fashion. Despite the primary role of lamina neurons in visual perception, it is still unclear how these neurons are specified from neuroepithelial (NE) progenitors. This study shows that a homothorax (hth) - eyes absent (eya) - sine oculis (so) - dachshund (dac) gene regulatory cassette is involved in this specification. Lamina neurons differentiate from NE progenitors that express hth, eya and so. One of the first events in the differentiation of lamina neurons is the upregulation of dac expression in response to Hh signaling. This dac upregulation, which marks the transition from NE progenitors into lamina precursors, also requires Eya/So, the expression of which is locked in by mutual feedback. dac expression is crucial for lamina differentiation because it ensures repression of hth, a negative regulator of single-minded, and thus dac allows further lamina neuron differentiation. Therefore, the specification of lamina neurons is controlled by coupling the cell-autonomous hth-eya-so-dac regulatory cassette to Hh signaling.

Jiang, N., Soba, P., Parker, E., Kim, C. C. and Parrish, J. Z. (2014). The microRNA bantam regulates a developmental transition in epithelial cells that restricts sensory dendrite growth. Development [Epub ahead of print]. PubMed ID: 24924190
Summary: As animals grow, many early born structures grow by cell expansion rather than cell addition; thus growth of distinct structures must be coordinated to maintain proportionality. This phenomenon is particularly widespread in the nervous system, with dendrite arbors of many neurons expanding in concert with their substrate to sustain connectivity and maintain receptive field coverage as animals grow. After rapidly growing to establish body wall coverage, dendrites of Drosophila class IV dendrite arborization (C4da) neurons grow synchronously with their substrate, the body wall epithelium, providing a system to study how proportionality is maintained during animal growth. This study shows that the microRNA bantam (ban) ensures coordinated growth of C4da dendrites and the epithelium through regulation of epithelial endoreplication, a modified cell cycle that entails genome amplification without cell division. In Drosophila larvae, epithelial endoreplication leads to progressive changes in dendrite-extracellular matrix (ECM) and dendrite-epithelium contacts, coupling dendrite/substrate expansion and restricting dendrite growth beyond established boundaries. Moreover, changes in epithelial expression of cell adhesion molecules, including the beta-integrin Myospheroid (Mys), accompany this developmental transition. Finally, endoreplication and the accompanying changes in epithelial mys expression are required to constrain late-stage dendrite growth and structural plasticity. Hence, modulating epithelium-ECM attachment probably influences substrate permissivity for dendrite growth and contributes to the dendrite-substrate coupling that ensures proportional expansion of the two cell types.

Okigawa, S., Mizoguchi, T., Okano, M., Tanaka, H., Isoda, M., Jiang, Y. J., Suster, M., Higashijima, S., Kawakami, K. and Itoh, M. (2014). Different combinations of Notch ligands and receptors regulate V2 interneuron progenitor proliferation and V2a/V2b cell fate determination. Dev Biol 391: 196-206. PubMed ID: 24768892
Summary: The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Using zebrafish embryos with altered Notch signaling, this study shows that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD (see Drosophila Delta), and three receptors, Notch1a, Notch1b, and Notch3 (see Drosophila Notch) redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib (see Drosophila Mind bomb), which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand-receptor combinations that occur concurrently during development in zebrafish.

Saturday, June 14th

Scopelliti, A., Cordero, J. B., Diao, F., Strathdee, K., White, B. H., Sansom, O. J. and Vidal, M. (2014). Local Control of Intestinal Stem Cell Homeostasis by Enteroendocrine Cells in the Adult Drosophila Midgut. Curr Biol 24(11): 1199-211. PubMed ID: 24814146
Summary: Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream. This paper reports a novel function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the Drosophila midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts - beyond its known roles in development - as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2 (Rickets), the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP. This study therefore has identified a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells.

Huo, Y. and Macara, I. G. (2014). The Par3-like polarity protein Par3L is essential for mammary stem cell maintenance. Nat Cell Biol 16: 529-537. PubMed ID: 24859006
Summary: The Par polarity proteins play key roles in asymmetric division of Drosophila melanogaster stem cells; however, whether the same mechanisms control stem cells in mammals is controversial. Although necessary for mammary gland morphogenesis, Par3 (Drosophila homolog: Bazooka) is not essential for mammary stem cell function. This study discovered that, instead, a previously uncharacterized protein, Par3-like (Par3L), is vital for mammary gland stem cell maintenance. Par3L function has been mysterious because, unlike Par3, it does not interact with atypical protein kinase C or the Par6 polarity protein. Par3L was found to be expressed by multipotent stem cells in the terminal end buds of murine mammary glands. Ablation of Par3L resulted in rapid and profound stem cell loss. Unexpectedly, Par3L, but not Par3, binds to the tumour suppressor protein Lkb1 (see Drosophila Lkb1) and inhibits its kinase activity. This interaction is key for the function of Par3L in mammary stem cell maintenance. These data reveal insights into a link between cell polarity proteins and stem cell survival, and uncover a biological function for Par3L.

Rogers, R. P. and Rogina, B. (2014). Increased mitochondrial biogenesis preserves intestinal stem cell homeostasis and contributes to longevity in Indy mutant flies. Aging (Albany NY) 6: 335-350. PubMed ID: 24827528
Summary: The Drosophila Indy (I'm not dead yet) gene encodes a plasma membrane transporter of Krebs cycle intermediates, with robust expression in tissues associated with metabolism. Reduced INDY alters metabolism and extends longevity in a manner similar to caloric restriction (CR); however, little is known about the tissue specific physiological effects of INDY reduction. This study focused on the effects of INDY reduction in the Drosophila midgut due to the importance of intestinal tissue homeostasis in healthy aging and longevity. The expression of Indy mRNA in the midgut changes in response to aging and nutrition. Genetic reduction of Indy expression increases midgut expression of the mitochondrial regulator spargel/dPGC-1, which is accompanied by increased mitochondrial biogenesis and reduced reactive oxygen species (ROS). These physiological changes in the Indy mutant midgut preserve intestinal stem cell (ISC) homeostasis and are associated with healthy aging. Genetic studies confirm that dPGC-1 mediates the regulatory effects of INDY, as illustrated by lack of longevity extension and ISC homeostasis in flies with mutations in both Indy and dPGC1. These data suggest INDY may be a physiological regulator that modulates intermediary metabolism in response to changes in nutrient availability and organismal needs by modulating dPGC-1.

Vogg, M. C., Owlarn, S., Perez Rico, Y. A., Xie, J., Suzuki, Y., Gentile, L., Wu, W. and Bartscherer, K. (2014). Stem cell-dependent formation of a functional anterior regeneration pole in planarians requires Zic and Forkhead transcription factors. Dev Biol 390: 136-148. PubMed ID: 24704339
Summary: Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. This study identified a zinc finger transcription factor, Smed-ZicA (see Drosophila Odd Paired)), as a downstream target of Smed-FoxD (see Drosophila Fork head), a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head - the anterior regeneration pole - and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. A model is suggested in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue.

Friday, June 13

Lin, Y. H., Chen, Y. C., Kao, T. Y., Lin, Y. C., Hsu, T. E., Wu, Y. C., Ja, W. W., Brummel, T. J., Kapahi, P., Yuh, C. H., Yu, L. K., Lin, Z. H., You, R. J., Jhong, Y. T. and Wang, H. D. (2014). Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans. Aging Cell [Epub ahead of print]. PubMed ID: 24889782
Summary: Target of rapamycin (TOR) signaling is a nutrient-sensing pathway controlling metabolism and lifespan. Although TOR signaling can be activated by a metabolite of diacylglycerol (DAG), phosphatidic acid (PA), the precise genetic mechanism through which DAG metabolism influences lifespan remains unknown. DAG is metabolized to either PA via the action of DAG kinase or 2-arachidonoyl-sn-glycerol by diacylglycerol lipase (DAGL). This study reports that in Drosophila and Caenorhabditis elegans, overexpression of diacylglycerol lipase (DAGL/inaE/dagl-1) or knockdown of diacylglycerol kinase (DGK/rdgA/dgk-5) extends lifespan and enhances response to oxidative stress. Phosphorylated S6 kinase (p-S6K) levels are reduced following these manipulations, implying the involvement of TOR signaling. Conversely, DAGL/inaE/dagl-1 mutants exhibit shortened lifespan, reduced tolerance to oxidative stress, and elevated levels of p-S6K. Additional results from genetic interaction studies are consistent with the hypothesis that DAG metabolism interacts with TOR and S6K signaling to affect longevity and oxidative stress resistance. These findings highlight conserved metabolic and genetic pathways that regulate aging.

Kon, N., Yoshikawa, T., Honma, S., Yamagata, Y., Yoshitane, H., Shimizu, K., Sugiyama, Y., Hara, C., Kameshita, I., Honma, K. and Fukada, Y. (2014). CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms. Genes Dev 28: 1101-1110. PubMed ID: 24831701
Summary: Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca(2+) in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. This study shows that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII; see Drosophila CamKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIalpha weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca(2+)-calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of CLOCK and BMAL1 (see Drosophila Clock and Cycle). These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities.

Fantauzzo, K. A. and Soriano, P. (2014). PI3K-mediated PDGFRalpha signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways. Genes Dev 28: 1005-1017. PubMed ID: 24788519
Summary: Previous studies have identified phosphatidylinositol 3-kinase (PI3K; see Drosophila Pi3K92E) as the main downstream effector of PDGFRalpha signaling during murine skeletal development. Autophosphorylation mutant knock-in embryos in which PDGFRalpha (See Drosophila Pvr) is unable to bind PI3K (Pdgfra(PI3K/PI3K)) exhibit skeletal defects affecting the palatal shelves, shoulder girdle, vertebrae, and sternum. To identify proteins phosphorylated by Akt (see Drosophila Akt1) downstream from PI3K-mediated PDGFRalpha signaling, Akt phosphorylation substrates from PDGF-AA-treated primary mouse embryonic palatal mesenchyme (MEPM) lysates were immunoprecipitated, and the peptides were analyzed by nanoliquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). This analysis generated a list of 56 proteins, including 10 that regulate cell survival and proliferation. It was demonstrated that MEPM cell survival is impaired in the presence of a PI3K inhibitor and that Pdgfra(PI3K/PI3K)-derived MEPMs do not proliferate in response to PDGF-AA treatment. Several of the identified Akt phosphorylation targets, including Ybox1, mediate cell survival through regulation of p53 (see Drosophila p53). Ybox1 binds both the p53 promoter and the p53 protein and expression of p53 is significantly decreased upon PDGF-AA treatment in MEPMs. Finally, this study demonstrated that introduction of a p53-null allele attenuates the vertebral defects found in Pdgfra(PI3K/PI3K) neonates. These findings identify p53 as a novel effector downstream from PI3K-engaged PDGFRalpha signaling that regulates survival and proliferation during skeletal development in vivo.

Bhandari, D., Raisch, T., Weichenrieder, O., Jonas, S. and Izaurralde, E. (2014). Structural basis for the Nanos-mediated recruitment of the CCR4-NOT complex and translational repression. Genes Dev 28: 888-901. PubMed ID: 24736845
Summary: The RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4-NOT deadenylase complex. This study shows that the three human Nanos paralogs (Nanos1-3) interact with the CNOT1 (see Drosophila Not1) C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1-3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1-3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. These findings provide the structural basis for the recruitment of the CCR4-NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4-NOT complex as the main effector complex for Nanos function.

Thursday, June 12th

Schwabe, T., Borycz, J. A., Meinertzhagen, I. A. and Clandinin, T. R. (2014). Differential Adhesion Determines the Organization of Synaptic Fascicles in the Drosophila Visual System. Curr Biol [Epub ahead of print]. PubMed ID: 24881879
Summary: Neuronal circuits in worms, flies, and mammals are organized so as to minimize wiring length for a functional number of synaptic connections, a phenomenon called wiring optimization. However, the molecular mechanisms that establish optimal wiring during development are unknown. This question was addressed by studying the role of N-cadherin in the development of optimally wired neurite fascicles in the peripheral visual system of Drosophila. Photoreceptor axons surround the dendrites of their postsynaptic targets, called lamina cells, within a concentric fascicle called a cartridge. N-cadherin is expressed at higher levels in lamina cells than in photoreceptors, and all genetic manipulations that invert these relative differences displace lamina cells to the periphery and relocate photoreceptor axon terminals into the center. It is concluded that Differential expression of a single cadherin is both necessary and sufficient to determine cartridge structure because it positions the most-adhesive elements that make the most synapses at the core and the less-adhesive elements that make fewer synapses at the periphery. These results suggest a general model by which differential adhesion can be utilized to determine the relative positions of axons and dendrites to establish optimal wiring.

Tanaka-Matakatsu, M., Miller, J., Borger, D., Tang, W. J. and Du, W. (2014). Daughterless homodimer synergizes with Eyeless to induce atonal expression and retinal neuron differentiation. Dev Biol [Epub ahead of print]. PubMed ID: 24886829
Summary: Class I Basic Helix-Loop-Helix (bHLH) transcription factors form homodimers or heterodimers with class II bHLH proteins. While bHLH heterodimers are known to have diverse roles, little is known about the role of class I homodimers. This study shows that a linked dimer of Daughterless (Da), the only Drosophila class I bHLH protein, activates Atonal (Ato) expression and retinal neuron differentiation synergistically with the retinal determination factor Eyeless (Ey). The HLH protein Extramacrocheate (Emc), which forms heterodimer with Da, antagonizes the synergistic activation from Da but not the Da-Da linked dimer with Ey. Da directly interacts with Ey and promotes Ey binding to the Ey binding site in the Ato 3' enhancer. Interestingly, the Ey binding site in the Ato 3' enhancer contains an embedded E-box that is also required for the synergistic activation by Ey and Da. Finally it was shown that mammalian homologs of Ey and Da can functionally replace their Drosophila counterparts to synergistically activate the Ato enhancer, suggesting that the observed function is evolutionary conserved.

Podufall, J., Tian, R., Knoche, E., Puchkov, D., Walter, A. M., Rosa, S., Quentin, C., Vukoja, A., Jung, N., Lampe, A., Wichmann, C., Bohme, M., Depner, H., Zhang, Y. Q., Schmoranzer, J., Sigrist, S. J. and Haucke, V. (2014). Presynaptic Role for the Cytomatrix Protein GIT in Synaptic Vesicle Recycling. Cell Rep [Epub ahead of print]. PubMed ID: 24882013
Summary: Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. This study identified the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and Stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.

Fernandes, V. M., Panchapakesan, S. S., Braid, L. R. and Verheyen, E. M. (2014). Nemo promotes Notch-mediated lateral inhibition downstream of proneural factors. Dev Biol [Epub ahead of print]. PubMed ID: 24880113
Summary: During neurogenesis, conserved tissue-specific proneural factors establish a cells competence to take on neural fate from within a field of unspecified cells. Proneural genes encode basic helix-loop-helix transcription factors that promote the expression of 'core' and subtype-specific target genes. Target genes include both pan-neuronal genes and genes that aid in the process of refinement, known as lateral inhibition. In this process, proneural gene expression is increased in the neural progenitor while simultaneously down-regulated in the surrounding cells, in a Notch signalling-dependent manner. This study identified nemo (nmo) as a target of members of both Drosophila Atonal and Achaete-Scute proneural factor families and found that mammalian proneural homologs induce Nemo-like-kinase (Nlk) expression in cell culture. nmo loss of function was found to lead to reduced expression of Notch targets and to perturbations in Notch-mediated lateral inhibition. Furthermore, Notch hyperactivity can compensate for nmo loss in the Drosophila eye. Thus nmo promotes Notch-mediated lateral inhibition downstream of proneural factors during neurogenesis.

Wednesday, June 11th

Wang, Z., Fan, M., Candas, D., Zhang, T. Q., Qin, L., Eldridge, A., Wachsmann-Hogiu, S., Ahmed, K. M., Chromy, B. A., Nantajit, D., Duru, N., He, F., Chen, M., Finkel, T., Weinstein, L. S. and Li, J. J. (2014). Cyclin B1/Cdk1 coordinates mitochondrial respiration for cell-cycle G2/M progression. Dev Cell 29: 217-232. PubMed ID: 24746669
Summary: A substantial amount of mitochondrial energy is required for cell-cycle progression. The mechanisms underlying the coordination of the mitochondrial respiration with cell-cycle progression, especially the G2/M transition, remain to be elucidated. This study shows that a fraction of cyclin B1/Cdk1 proteins (see Drosophila Cyclin B and Cdc2) localizes to the matrix of mitochondria and phosphorylates a cluster of mitochondrial proteins, including the complex I (CI) subunits in the respiratory chain. Cyclin B1/Cdk1-mediated CI phosphorylation enhances CI activity, whereas deficiency of such phosphorylation in each of the relevant CI subunits results in impairment of CI function. Mitochondria-targeted cyclin B1/Cdk1 increases mitochondrial respiration with enhanced oxygen consumption and ATP generation, which provides cells with efficient bioenergy for G2/M transition and shortens overall cell-cycle time. Thus, cyclin B1/Cdk1-mediated phosphorylation of mitochondrial substrates allows cells to sense and respond to increased energy demand for G2/M transition and, subsequently, to upregulate mitochondrial respiration for successful cell-cycle progression.

Li, D., Sakuma, R., Vakili, N. A., Mo, R., Puviindran, V., Deimling, S., Zhang, X., Hopyan, S. and Hui, C. C. (2014). Formation of proximal and anterior limb skeleton requires early function of irx3 and irx5 and is negatively regulated by shh signaling. Dev Cell 29: 233-240. PubMed ID: 24726282
Summary: Limb skeletal pattern relies heavily on graded Sonic hedgehog (Shh) signaling. As a morphogen and growth cue, Shh regulates identities of posterior limb elements, including the ulna/fibula and digits 2 through 5. In contrast, proximal and anterior structures, including the humerus/femur, radius/tibia, and digit 1, are regarded as Shh independent, and mechanisms governing their specification are unclear. This study shows that patterning of the proximal and anterior limb skeleton involves two phases. Irx3 and Irx5 (Irx3/5; see Drosophila Mirror) are essential in the initiating limb bud to specify progenitors of the femur, tibia, and digit 1. However, these skeletal elements can be restored in Irx3/5 null mice when Shh signaling is diminished, indicating that Shh negatively regulates their formation after initiation. These data provide genetic evidence supporting the concept of early specification and progressive determination of anterior limb pattern.

Zhulyn, O., Li, D., Deimling, S., Vakili, N. A., Mo, R., Puviindran, V., Chen, M. H., Chuang, P. T., Hopyan, S. and Hui, C. C. (2014). A switch from low to high shh activity regulates establishment of limb progenitors and signaling centers. Dev Cell 29: 241-249. PubMed ID: 24726283
Summary: The patterning and growth of the embryonic vertebrate limb is dependent on Sonic hedgehog (Shh; see Drosophila Hedgehog), a morphogen that regulates the activity of Gli transcription factors (see Drosophila Cubitus interuptus). However, Shh expression is not observed during the first 12 hr of limb development. During this phase, the limb bud is prepatterned into anterior and posterior regions through the antagonistic actions of transcription factors Gli3 and Hand2 (see Drosophila Hand). This study demonstrates that precocious activation of Shh signaling during this early phase interferes with the Gli3-dependent specification of anterior progenitors, disturbing establishment of signaling centers and normal outgrowth of the limb. These findings illustrate that limb development requires a sweet spot in the level and timing of pathway activation that allows for the Shh-dependent expansion of posterior progenitors without interfering with early prepatterning functions of Gli3/Gli3R or specification of anterior progenitors.

Smykal, V., Daimon, T., Kayukawa, T., Takaki, K., Shinoda, T. and Jindra, M. (2014). Importance of juvenile hormone signaling arises with competence of insect larvae to metamorphose. Dev Biol 390: 221-230. PubMed ID: 24662045
Summary: Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori, were chosed. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH, were examined. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, it is proposed that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth.

Tuesday, June 10th

Petkova, M. D., Little, S. C., Liu, F. and Gregor, T. (2014). Maternal Origins of Developmental Reproducibility. Curr Biol [Epub ahead of print]. PubMed ID: 24856210
Summary: Cell fate decisions during multicellular development are precisely coordinated, leading to highly reproducible macroscopic structural outcomes. The origins of this reproducibility are found at the molecular level during the earliest stages of development when patterns of morphogen molecules emerge reproducibly. However, although the initial conditions for these early stages are determined by the female during oogenesis, it is unknown whether reproducibility is perpetuated from oogenesis or reacquired by the zygote. To address this issue in the early Drosophila embryo, this study sought to count individual maternally deposited bicoid mRNA molecules and compare variability between embryos with previously observed fluctuations in the Bicoid protein gradient. Independent methods were developed to quantify total amounts of mRNA in individual embryos and show that mRNA counts are highly reproducible between embryos to within approximately 9%, matching the reproducibility of the protein gradient. Reproducibility emerges from perfectly linear feedforward processes: changing the genetic dosage in the female leads to proportional changes in both mRNA and protein numbers in the embryo. These results indicate that the reproducibility of the morphological structures of embryos originates during oogenesis, which is when the expression of maternally provided patterning factors is precisely controlled.

Liu, W. J., Reece-Hoyes, J. S., Walhout, A. J. and Eisenmann, D. M. (2014). Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6. BMC Dev Biol 14: 17. PubMed ID: 24885717
Summary: Hox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 (homolog of Drosophila Sex-combs reduced) is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, transcription factors necessary for lin-39 expression in the VPCs, factors were sought that initiate lin-39 expression in the embryo. The yeast one-hybrid (Y1H) method was used to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and several factors were characterized by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations inlin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site that was identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs. These results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintains lin-39 expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression in C. elegans is complicated, redundant and robust.

Kronja, I., Yuan, B., Eichhorn, S. W., Dzeyk, K., Krijgsveld, J., Bartel, D. P. and Orr-Weaver, T. L. (2014). Widespread Changes in the Posttranscriptional Landscape at the Drosophila Oocyte-to-Embryo Transition. Cell Rep [Epub ahead of print]. PubMed ID: 24882012
Summary: The oocyte-to-embryo transition marks the onset of development. The initial phase of this profound change from the differentiated oocyte to the totipotent embryo occurs in the absence of both transcription and mRNA degradation. This study combined global polysome profiling, ribosome-footprint profiling, and quantitative mass spectrometry in a comprehensive approach to delineate the translational and proteomic changes that occur during this important transition in Drosophila. The results show that Pan gu (PNG) kinase is a critical regulator of the extensive changes in the translatome, acting uniquely at this developmental window. Analysis of the proteome in png) mutants provided insights into the contributions of translation to changes in protein levels, revealing a compensatory dynamic between translation and protein turnover during proteome remodeling at the return to totipotency. The proteome changes additionally suggested regulators of meiosis and early embryogenesis, including the conserved H3K4 demethylase LID, which was demonstrated to be required during this period despite transcriptional inactivity.

Wisotzkey, R. G., Quijano, J. C., Stinchfield, M. J. and Newfeld, S. J. (2014). New gene evolution in the Bonus-TIF1-gamma/TRIM33 family impacted the architecture of the vertebrate dorsal-ventral patterning network. Mol Biol Evol. PubMed ID: 24881051
Summary: Uncovering how a new gene acquires its function and understanding how the function of a new gene influences existing genetic networks are important topics in evolutionary biology. This study demonstrated nonconservation for the embryonic functions of Drosophila Bonus and its newest vertebrate relative TIF1-gamma/TRIM33. It was shown previously that TIF1-gamma/TRIM33 functions as an ubiquitin ligase for the Smad4 (see Drosophila Medea) signal transducer and antagonizes the Bone Morphogenetic Protein (BMP) signaling network underlying vertebrate dorsal-ventral axis formation. This study showed that Bonus functions as an agonist of the Decapentaplegic (Dpp) signaling network underlying dorsal-ventral axis formation in flies. The absence of conservation for the roles of Bonus and TIF1-gamma/TRIM33 reveals a shift in the dorsal-ventral patterning networks of flies and mice, systems that were previously considered wholly conserved. The shift occurred when the new gene TIF1-gamma/TRIM33 replaced the function of the ubiquitin ligase Nedd4L in the lineage leading to vertebrates. Evidence of this replacement is the demonstration that Nedd4 performs the function of TIF1-gamma/TRIM33 in flies during dorsal-ventral axis formation. The replacement allowed vertebrate Nedd4L to acquire novel functions as a ubiquitin ligase of vertebrate-specific Smad proteins. Overall this data reveals that the architecture of the Dpp/BMP dorsal-ventral patterning network continued to evolve in the vertebrate lineage, after separation from flies, via the incorporation of new genes.

Monday, June 9th

Ostojic, I., Boll, W., Waterson, M. J., Chan, T., Chandra, R., Pletcher, S. D. and Alcedo, J. (2014). Positive and negative gustatory inputs affect Drosophila lifespan partly in parallel to dFOXO signaling. Proc Natl Acad Sci U S A. PubMed ID: 24847072
Summary: In Caenorhabditis elegans, a subset of gustatory neurons, as well as olfactory neurons, shortens lifespan, whereas a different subset of gustatory neurons lengthens it. Recently, the lifespan-shortening effect of olfactory neurons has been reported to be conserved in Drosophila. The Drosophila gustatory system also affects lifespan in a bidirectional manner. Taste inputs were found to shorten lifespan through inhibition of the insulin pathway effector dFOXO, whereas other taste inputs lengthen lifespan in parallel to this pathway. It was also noted that the gustatory influence on lifespan does not necessarily depend on food intake levels. Finally, the nature of some of the taste inputs that could shorten versus lengthen lifespan was identified. Together these data suggest that different gustatory cues can modulate the activities of distinct signaling pathways, including different insulin-like peptides, to promote physiological changes that ultimately affect lifespan.

Yu, J., Chia, J., Canning, C. A., Jones, C. M., Bard, F. A. and Virshup, D. M. (2014). WLS Retrograde Transport to the Endoplasmic Reticulum during Wnt Secretion. Dev Cell 29: 277-291. PubMed ID: 24768165
Summary: Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. This study found that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. An ER-targeting sequence was identified at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle.

Yau, R. G., Peng, Y., Valiathan, R. R., Birkeland, S. R., Wilson, T. E. and Weisman, L. S. (2014). Release from myosin V via regulated recruitment of an E3 ubiquitin ligase controls organelle localization. Dev Cell 28: 520-533. PubMed ID: 24636257
Summary: Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin V, Myo2 (see Drosophila Didum), binds the vacuole-specific adaptor Vac17 to transport the vacuole from the mother cell to the bud. This study shows that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 that recruits the E3 ligase Dma1 to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, it was found that the termination of peroxisome transport also requires Dma1. It is predicted that this is a general mechanism that detaches myosin V from select cargoes.

Mahesh, G., Jeong, E., Ng, F. S., Liu, Y., Gunawardhana, K., Houl, J. H., Yildirim, E., Amunugama, R., Jones, R., Allen, D. L., Edery, I., Kim, E. Y. and Hardin, P. E. (2014). Phosphorylation of the Transcription Activator CLOCK Regulates Progression Through a ~24h Feedback Loop to Influence Circadian Period in Drosophila. J Biol Chem [Epub ahead of print]. PubMed ID: 24872414
Summary: Circadian (~24hr) clocks control daily rhythms in metabolism, physiology and behavior in animals, plants and microbes. In Drosophila, these clocks keep circadian time via transcriptional feedback loops in which Clock-Cycle (Clk-Cyc) initiates transcription of period (per) and timeless (tim), accumulating levels of Per and Tim feed back to inhibit Clk-Cyc, and degradation of Per and Tim allows Clk-Cyc to initate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in Per and Clk phosphorylation, where Per and Clk phosphorylation is high during transcriptional repression. Per phosphorylation at specific sites controls its subcellular localization, activity and stability, but comparatively little is known about the identity and function of Clk phosphorylation sites. This study identified eight Clk phosphorylation sites via mass spectrometry and determined how phosphorylation at these sites impacts behavioral and molecular rhythms by transgenic rescue of a new Clk null mutant. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten circadian period, whereas loss of Clk phosphorylation at serine 859 increases Clk activity, thereby increasing Per levels and accelerating transcriptional repression. These results demonstrate that Clk phosphorylation influences circadian period by regulating Clk activity and progression through the feedback loop.

Sunday, June 8th

Cordero, J. B., Ridgway, R. A., Valeri, N., Nixon, C., Frame, M. C., Muller, W. J., Vidal, M. and Sansom, O. J. (2014). c-Src drives intestinal regeneration and transformation. EMBO J [Epub ahead of print]. PubMed ID: 24788409
Summary: The non-receptor tyrosine kinase c-Src, hereafter referred to as Src, is overexpressed or activated in multiple human malignancies. There has been much speculation about the functional role of Src in colorectal cancer (CRC), with Src amplification and potential activating mutations in up to 20% of the human tumours, although this has never been addressed due to multiple redundant family members. This study used the adult Drosophila and mouse intestinal epithelium as paradigms to define a role for Src during tissue homeostasis, damage-induced regeneration and hyperplasia. Through genetic gain and loss of function experiments, it was demonstrated that Src is necessary and sufficient to drive intestinal stem cell (ISC) proliferation during tissue self-renewal, regeneration and tumourigenesis. Surprisingly, Src plays a non-redundant role in the mouse intestine, which cannot be substituted by the other family kinases Fyn and Yes. Mechanistically, it was shown that Src drives ISC proliferation through upregulation of EGFR and activation of Ras/MAPK and Stat3 signalling. Therefore, this study demonstrated a novel essential role for Src in intestinal stem/progenitor cell proliferation and tumourigenesis initiation in vivo.

Geng, J., Xia, L., Li, W. and Dou, F. (2014). The C-Terminus of Tau protein plays an important role in its stability and toxicity. J Mol Neurosci [Epub ahead of print]. PubMed ID: 24788226
Summary: The identification of tau fragments generated by proteolysis in the neurons of AD patients and in neurofibrillary tangles encourages research on the toxicity of truncated tau. However, the detailed mechanism underlying the proteolysis-induced toxicity of tau is not clear and even controversial in some cases. The study used Drosophila as a model to evaluate the toxicity of a set of truncated tau fragments in vivo and found that the flies harboring C-terminal-truncated tau exhibited less toxicity due to the unstable characteristic of C-terminal-truncated tau fragments. Further study carried out in cultured Drosophila Kc cells revealed that C-terminal-truncated tau fragments degrade faster than full-length tau or N-terminal-truncated fragments. Collectively, these data implicate proteolysis of tau as an important pathway mediating tau degradation and neurotoxicity in vivo.

Schutte, R. J., Schutte, S. S., Algara, J., Barragan, E. V., Gilligan, J., Staber, C., Savva, Y. A., Smith, M. A., Reenan, R. and O'Dowd, D. K. (2014). Knock-in model of Dravet Syndrome reveals a constitutive and conditional reduction in sodium current. J Neurophysiol [Epub ahead of print]. PubMed ID: 24805083
Summary: Hundreds of mutations in the SCN1A sodium channel gene confer a wide spectrum of epileptic disorders, requiring efficient model systems to study cellular mechanisms and identify potential therapeutic targets. It has been demonstrated that Drosophila knock-in flies carrying a Generalized epilepsy with febrile seizures plus (GEFS+) causing SCN1A mutation (K1270T) results in a primarily conditional increase in sodium current activity that contributes to a heat-induced seizure phenotype. To determine whether different SCN1A mutations cause distinct phenotypes in Drosophila as they do in humans, this paper focuses on a knock-in line carrying a mutation that causes a more severe seizure disorder, Dravet Syndrome (DS), in humans. Introduction of the DS SCN1A mutation (S1231R) into the Drosophila sodium channel gene, para, results in flies that exhibit spontaneous and heat-induced seizures with distinct characteristics and lower onset temperature than the GEFS+ flies. Electrophysiological studies of GABAergic interneurons in the brains of adult DS flies reveal, for the first time in an in vivo model system, that a missense DS mutation causes a constitutive and conditional reduction in sodium current activity and repetitive firing. In addition, feeding with the serotonin precursor 5-HTP suppresses heat-induced seizures in DS but not GEFS+ flies. The distinct alterations of sodium currents in DS and GEFS+ GABAergic interneurons demonstrate that both loss- and gain-of-function alterations in sodium currents are capable of causing reduced repetitive firing and seizure phenotypes. The mutation-specific effects of 5-HTP on heat-induced seizures suggest the serotonin pathway as a potential therapeutic target for DS.

Thomas, R. E., Andrews, L. A., Burman, J. L., Lin, W. Y. and Pallanck, L. J. (2014). PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix. PLoS Genet 10: e1004279. PubMed ID: 24874806
Summary: Loss-of-function mutations in PINK1 (see Drosophila Pink1), which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson's disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, a screen was performed for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. Genetic perturbations targeting the matrix-localized protease Lon were found to cause dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPRmt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPRmt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. These findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria.

Cornelissen, T., Haddad, D., Wauters, F., Van Humbeeck, C., Mandemakers, W., Koentjoro, B., Sue, C., Gevaert, K., De Strooper, B., Verstreken, P. and Vandenberghe, W. (2014). The deubiquitinase USP15 antagonizes Parkin-mediated mitochondrial ubiquitination and mitophagy. Hum Mol Genet [Epub ahead of print]. PubMed ID: 24852371
Summary: Loss-of-function mutations in PARK2, the gene encoding the E3 ubiquitin ligase Parkin, are the most frequent cause of recessive Parkinson's disease (PD). Parkin translocates from the cytosol to depolarized mitochondria, ubiquitinates outer mitochondrial membrane proteins and induces selective autophagy of the damaged mitochondria (mitophagy). This study shows that Ubiquitin-specific protease 15 (USP15), a deubiquitinating enzyme (DUB) widely expressed in brain and other organs, opposes Parkin-mediated mitophagy, while a panel of other DUBs and a catalytically inactive version of USP15 do not. Moreover, knockdown of USP15 rescues the mitophagy defect of PD patient fibroblasts with PARK2 mutations and decreased Parkin levels. USP15 does not affect the ubiquitination status of Parkin or Parkin translocation to mitochondria, but counteracts Parkin-mediated mitochondrial ubiquitination. Knockdown of the DUB CG8334, the closest homolog of USP15 in Drosophila, largely rescues the mitochondrial and behavioural defects of parkin RNAi flies. These data identify USP15 as an antagonist of Parkin and suggest that USP15 inhibition could be a therapeutic strategy for PD cases caused by reduced Parkin levels.

Saturday, June 7th

Xu, S., Tyagi, S. and Schedl, P. (2014). Spermatid Cyst Polarization in Drosophila Depends upon apkc and the CPEB Family Translational Regulator orb2. PLoS Genet 10: e1004380. PubMed ID: 24830287
Summary: Mature Drosophila sperm are highly polarized cells - on one side is a nearly 2 mm long flagellar tail that comprises most of the cell, while on the other is the sperm head, which carries the gamete's genetic information. The polarization of the sperm cells commences after meiosis is complete and the 64-cell spermatid cyst begins the process of differentiation. The spermatid nuclei cluster to one side of the cyst, while the flagellar axonemes grows from the other. The elongating spermatid bundles are also polarized with respect to the main axis of the testis; the sperm heads are always oriented basally, while the growing tails extend apically. This orientation within the testes is important for transferring the mature sperm into the seminal vesicles. Orienting cyst polarization with respect to the main axis of the testis is shown in this study to depend upon atypical Protein Kinase C (aPKC), a factor implicated in polarity decisions in many different biological contexts. When apkc activity is compromised in the male germline, the direction of cyst polarization within this organ is randomized. Significantly, the mechanisms used to spatially restrict apkc activity to the apical side of the spermatid cyst are different from the canonical cross-regulatory interactions between this kinase and other cell polarity proteins that normally orchestrate polarization. The asymmetric accumulation of aPKC protein in the cyst is shown to depend on an mRNA localization pathway that is regulated by the Drosophila CPEB protein Orb2. orb2 is required to properly localize and activate the translation of apkc mRNAs in polarizing spermatid cysts. It was also shown that orb2 functions not only in orienting cyst polarization with respect to the apical-basal axis of the testis, but also in the process of polarization itself. One of the orb2 targets in this process is its own mRNA. Moreover, the proper execution of this orb2 autoregulatory pathway depends upon apkc.

Cai, D., Chen, S. C., Prasad, M., He, L., Wang, X., Choesmel-Cadamuro, V., Sawyer, J. K., Danuser, G. and Montell, D. J. (2014). Mechanical Feedback through E-Cadherin Promotes Direction Sensing during Collective Cell Migration. Cell 157: 1146-1159. PubMed ID: 24855950
Summary: E-cadherin is a major homophilic cell-cell adhesion molecule that inhibits motility of individual cells on matrix. However, its contribution to migration of cells through cell-rich tissues is less clear. An in vivo sensor of mechanical tension across E-cadherin molecules has been developed, which was combined with cell-type-specific RNAi, photoactivatable Rac, and morphodynamic profiling, to interrogate how E-cadherin contributes to collective migration of cells between other cells. Using the Drosophila ovary as a model, it was found that adhesion between border cells and their substrate, the nurse cells, functions in a positive feedback loop with Rac and actin assembly to stabilize forward-directed protrusion and directionally persistent movement. Adhesion between individual border cells communicates direction from the lead cell to the followers. Adhesion between motile cells and polar cells holds the cluster together and polarizes each individual cell. Thus, E-cadherin is an integral component of the guidance mechanisms that orchestrate collective chemotaxis in vivo.

Jia, D., Tamori, Y., Pyrowolakis, G. and Deng, W. M. (2014). Regulation of broad by the Notch pathway affects timing of follicle cell development. Dev Biol [Epub ahead of print]. PubMed ID: 24815210
Summary: During Drosophila oogenesis, activation of Notch signaling in the follicular epithelium (FE) around stage 6 of oogenesis is essential for entry into the endocycle and a series of other changes such as cell differentiation and migration of subsets of the follicle cells. Notch induces the expression of zinc finger protein Hindsight and suppresses homeodomain protein Cut to regulate the mitotic/endocycle (ME) switch. This study reports that broad (br), encoding a small group of zinc-finger transcription factors resulting from alternative splicing, is a transcriptional target of Notch nuclear effector Suppressor of Hairless (Su(H)). The early pattern of Br in the FE, uniformly expressed except in the polar cells, is established by Notch signaling around stage 6, through the binding of Su(H) to the br early enhancer (brE) region. Mutation of the Su(H) binding site leads to a significant reduction ofbrE reporter expression in follicle cells undergoing the endocycle. Chromatin immunoprecipitation results further confirm Su(H) binding to the br early enhancer. Consistent with its expression in follicle cells during midoogenesis, loss of br function results in a delayed entry into the endocycle. These findings suggest an important role of br in the timing of follicle cell development, and its transcriptional regulation by the Notch pathway.

Furriols, M. and Casanova, J. (2014). Germline and somatic vitelline proteins colocalize in aggregates in the follicular epithelium of Drosophila ovaries. Fly (Austin) 8 [Epub ahead of print]. PubMed ID: 24813257
Summary: Nasrat and Polehole, two Drosophila proteins related functionally and by sequence, are secreted from the oocyte and incorporated into the vitelline membrane, where they play a role in the integrity of the same and in the activation of embryonic Torso RTK. In addition, they also accumulate in a punctate pattern in the follicular epithelium. This study shows that their accumulation at the follicle cells depends on their gene expression in the germline, indicating that these proteins move from the oocyte to the follicle cells in a process that does not require endocytosis. Finally cell markers were used to examine the distribution of these proteins at the follicle cells, and they were shown to accumulate in aggregates with vitelline membrane proteins in close association with the plasmatic membrane. It is proposed that these aggregates represent spatially restricted sinks for vitelline membrane proteins that fail to be incorporated into vitelline bodies and later on into the vitelline membrane.

Herzig, B., Yakulov, T. A., Klinge, K., Gunesdogan, U., Jackle, H. and Herzig, A. (2014). Bällchen is required for self-renewal of germline stem cells in Drosophila melanogaster. Biol Open [Epub ahead of print]. PubMed ID: 24876388
Summary: Self-renewing stem cells are pools of undifferentiated cells, which are maintained in cellular niche environments by distinct tissue-specific signalling pathways. In Drosophila melanogaster, female germline stem cells (GSCs) are maintained in a somatic niche of the gonads by BMP signalling. This study reports a novel function of the Drosophila kinase Bällchen (BALL), showing that its cell autonomous role is to maintain the self-renewing capacity of female GSCs independent of BMP signalling. ball mutant GSCs are eliminated from the niche and subsequently differentiate into mature eggs, indicating that BALL is largely dispensable for differentiation. Similar to female GSCs, BALL is required to maintain self-renewal of male GSCs, suggesting a tissue independent requirement of BALL for self-renewal of germline stem cells.

Friday, June 6th

Kassavetis, G. A. and Kadonaga, J. T. (2014). The Annealing Helicase and Branch Migration Activities of Drosophila HARP. PLoS One 9: e98173. PubMed ID: 24866343
Summary: HARP (SMARCAL1, MARCAL1) is an annealing helicase that functions in the repair and restart of damaged DNA replication forks through its DNA branch migration and replication fork regression activities. HARP is conserved among metazoans. HARP from invertebrates differs by the absence of one of the two HARP-specific domain repeats found in vertebrates. The annealing helicase and branch migration activity of invertebrate HARP has not been documented. This study found that HARP from Drosophila melanogaster retains the annealing helicase activity of human HARP, the ability to disrupt D-loops and to branch migrate Holliday junctions, but fails to regress model DNA replication fork structures. A comparison of human and Drosophila HARP on additional substrates revealed that both HARPs are competent in branch migrating a bidirectional replication bubble composed of either DNA:DNA or RNA:DNA hybrid. Human, but not Drosophila, HARP is also capable of regressing a replication fork structure containing a highly stable poly rG:dC hybrid. Persistent RNA:DNA hybrids in vivo can lead to replication fork arrest and genome instability. The ability of HARP to strand transfer hybrids may signify a hybrid removal function for this enzyme, in vivo.

Basquin, D., Spierer, A., Begeot, F., Koryakov, D. E., Todeschini, A. L., Ronsseray, S., Vieira, C., Spierer, P. and Delattre, M. (2014). The Drosophila Su(var)3-7 Gene Is Required for Oogenesis and Female Fertility, Genetically Interacts with piwi and aubergine, but Impacts Only Weakly Transposon Silencing. PLoS One 9: e96802. PubMed ID: 24820312
Summary: Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. This study analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. Evidence is presented that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead to a proposal that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.

Dorogova, N. V., Fedorova, E. V., Bolobolova, E., Ogienko, A. A. and Baricheva, E. M. (2014). GAGA protein is essential for male germ cell development in Drosophila. Genesis [Epub ahead of print]. PubMed ID: 24817547
Summary: The Drosophila Trithorax-like (Trl) gene encodes a GAGA factor which regulates a number of developmentally important genes. This study identified a new function for Drosophila GAGA factor in male germ cell development. Trl mutants carrying strong hypomorphic alleles display loss of primordial germ cells during their migration in embryogenesis and severe disruption in mitochondria structure during early spermatogenesis. The mutation resulted in small testes formation, a deficit of germ cells, abnormal mitochondrial morphogenesis, spermatocyte death through autophagy and partial or complete male sterility. Pleiotropic mutation effects can be explained by the misexpression of GAGA factor target genes, the products of which are required for germ cell progression into mature sperm.

Sorourian, M., Kunte, M. M., Domingues, S., Gallach, M., Ozdil, F., Rio, J. and Betran, E. (2014). Relocation Facilitates the Acquisition of Short cis-Regulatory Regions that Drive the Expression of Retrogenes during Spermatogenesis in Drosophila. Mol Biol Evol [Epub ahead of print]. PubMed ID: 24855141
Summary: Retrogenes are functional processed copies of genes that originate via the retrotranscription of a mRNA intermediate and often exhibit testis-specific expression. Although this expression pattern appears to be favored by selection, the origin of such expression bias remains unexplained. The regulation of two young testis-specific Drosophila retrogenes, Dntf-2r and Pros28.1A, was studied using genetic transformation and the EGFP reporter gene in D. melanogaster. Two different short (<24 bp) regions upstream of the transcription start sites (TSSs) act as testis-specific regulatory motifs in these genes. The Dntf-2r regulatory region is similar to the known β2 tubulin 14 bp testis motif (β2-UE1). Comparative sequence analyses reveal that this motif was already present before the Dntf-2r insertion and was likely driving the transcription of a non-coding RNA. It was also shown that the β2-UE1 occurs in the regulatory regions of other testis-specific retrogenes, and is functional in either orientation. In contrast, the Pros28.1A testes regulatory region in D. melanogaster appears to be novel. Only Pros28.1B, an older paralog of the Pros28.1 gene family, seems to carry a similar regulatory sequence. It is unclear how the Pros28.1A regulatory region was acquired in D. melanogaster, but it might have evolved de novo from within a region that may have been pre-primed for testes expression. We conclude that relocation is critical for the evolutionary origin of male germline-specific cis-regulatory regions of retrogenes because expression depends on either the site of the retrogene insertion or on sequence changes close to the TSS thereafter. As a consequence we infer that positive selection will play a role in the evolution of these regulatory regions and can often act from the moment of the retrocopy insertion.

Thursday, June 5th

Novak, Z. A., Conduit, P. T., Wainman, A. and Raff, J. W. (2014). Asterless Licenses Daughter Centrioles to Duplicate for the First Time in Drosophila Embryos. Curr Biol [Epub ahead of print]. PubMed ID: 24835456
Summary: Centrioles form centrosomes and cilia, and defects in any of these three organelles are associated with human disease. Centrioles duplicate once per cell cycle, when a mother centriole assembles an adjacent daughter during S phase. Daughter centrioles cannot support the assembly of another daughter until they mature into mothers during the next cell cycle. The molecular nature of this daughter-to-mother transition remains mysterious. Pioneering studies in C. elegans identified a set of core proteins essential for centriole duplication, and a similar set have now been identified in other species. The protein kinase ZYG-1/Sak/Plk4 recruits the inner centriole cartwheel components SAS-6 and SAS-5/Ana2/STIL, which then recruit SAS-4/CPAP, which in turn helps assemble the outer centriole microtubules. In flies and humans, the Asterless/Cep152 protein interacts with Sak/Plk4 and Sas-4/CPAP and is required for centriole duplication, although its precise role in the assembly pathway is unclear. This study shows that Asl is not incorporated into daughter centrioles as they assemble during S phase but is only incorporated once mother and daughter separate at the end of mitosis. The initial incorporation of Asterless (Asl) is irreversible, requires DSas-4, and, crucially, is essential for daughter centrioles to mature into mothers that can support centriole duplication. Therefore a 'dual-licensing' model of centriole duplication is proposed, in which Asl incorporation provides a permanent primary license to allow new centrioles to duplicate for the first time, while centriole disengagement provides a reduplication license to allow mother centrioles to duplicate again.

Rubin, T., Karess, R. E. and Rahmani, Z. (2014). Cenp-meta is required for sustained spindle checkpoint. Biol Open. PubMed ID: 24876387
Summary: Cenp-E is a kinesin-like motor protein required for efficient end-on attachment of kinetochores to the spindle microtubules. Cenp-E immunodepletion in Xenopus mitotic extracts results in the loss of mitotic arrest and massive chromosome missegregation, whereas its depletion in mammalian cells leads to chromosome segregation defects despite the presence of a functional spindle assembly checkpoint (SAC). Cenp-meta has previously been reported to be the Drosophila homolog of vertebrate Cenp-E. In this study, it was shown that cenp-metaΔ mutant neuroblasts arrest in mitosis when treated with colchicine. cenp-metaΔ mutant cells display a mitotic delay. Yet, despite the persistence of the two checkpoint proteins Mad2 and BubR1 on unattached kinetochores, these cells eventually enter anaphase and give rise to highly aneuploid daughter cells. Indeed, it was found that cenp-metaΔ mutant cells display a slow but continuous degradation of cyclin B, which eventually triggers the mitotic exit observed. Thus, these data provide evidence for a role of Cenp-meta in sustaining the SAC response.

Wan, L., Tan, M., Yang, J., Inuzuka, H., Dai, X., Wu, T., Liu, J., Shaik, S., Chen, G., Deng, J., Malumbres, M., Letai, A., Kirschner, M. W., Sun, Y. and Wei, W. (2014). APC(Cdc20) Suppresses Apoptosis through Targeting Bim for Ubiquitination and Destruction. Dev Cell 29: 377-391. PubMed ID: 24871945
Summary: Anaphase-promoting complex Cdc20 (APCCdc20; Fizzy in Drosophila) plays pivotal roles in governing mitotic progression. By suppressing APCCdc20, antimitotic agents activate the spindle-assembly checkpoint and induce apoptosis after prolonged treatment, whereas depleting endogenous Cdc20 suppresses tumorigenesis in part by triggering mitotic arrest and subsequent apoptosis. However, the molecular mechanism(s) underlying apoptosis induced by Cdc20 abrogation remains poorly understood. This study reports the BH3-only proapoptotic protein BCL2-like 11 (Bim) as an APCCdc20 target, such that depletion of Cdc20 sensitizes cells to apoptotic stimuli. Strikingly, Cdc20 and multiple APC-core components were identified in a small interfering RNA screen that, upon knockdown, sensitizes otherwise resistant cancer cells to chemoradiation in a Bim-dependent manner. Consistently, human adult T cell leukemia cells that acquire elevated APCCdc20 activity via expressing the Tax viral oncoprotein exhibit reduced Bim levels and resistance to anticancer agents. These results reveal an important role for APCCdc20 in governing apoptosis, strengthening the rationale for developing specific Cdc20 inhibitors as effective anticancer agents.

Hehnly, H. and Doxsey, S. (2014). Rab11 endosomes contribute to mitotic spindle organization and orientation. PubMed ID: 24561039
Summary: During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 endosomes in mitosis. This study, carried out in cultured mammalian cells, shows that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. Mitotic recycling endosomes were shown to bind gamma-TuRC components and associate with tubulin in vitro. Rab11 depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively active Rab11 increases astral microtubules, restores gamma-tubulin spindle pole localization, and generates robust spindles. This suggests a role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. It is proposed that Rab11 and its associated endosomes cocontribute to these processes through retrograde transport to poles by dynein.

Wednesday, June 4th

Kuert, P. A., Hartenstein, V., Bello, B. C., Lovick, J. K. and Reichert, H. (2014). Neuroblast lineage identification and lineage-specific Hox gene action during postembryonic development of the subesophageal ganglion in the Drosophila central brain. Dev Biol 390: 102-115. PubMed ID: 24713419
Summary: The central brain of Drosophila consists of the supraesophageal ganglion (SPG) and the subesophageal ganglion (SEG), both of which are generated by neural stem cell-like neuroblasts during embryonic and postembryonic development. Considerable information has been obtained on postembryonic development of the neuroblasts and their lineages in the SPG. In contrast, very little is known about neuroblasts, neural lineages, or any other aspect of the postembryonic development in the SEG. This study characterized the neuroanatomy of the larval SEG in terms of tracts, commissures, and other landmark features as compared to a thoracic ganglion. Then clonal MARCM labeling was used to identify all adult-specific neuroblast lineages in the late larval SEG, and a surprisingly small number of neuroblast lineages, 13 paired and one unpaired, were found. The Hox genes Dfd, Scr, and Antp are expressed in a lineage-specific manner in these lineages during postembryonic development. Hox gene loss-of-function causes lineage-specific defects in axonal targeting and reduction in neural cell numbers. Moreover, it results in the formation of novel ectopic neuroblast lineages. Apoptosis block also results in ectopic lineages suggesting that Hox genes are required for lineage-specific termination of proliferation through programmed cell death. Taken together, these findings show that postembryonic development in the SEG is mediated by a surprisingly small set of identified lineages and requires lineage-specific Hox gene action to ensure the correct formation of adult-specific neurons in the Drosophila brain.

Shieh, B. H., Kristaponyte, I. and Hong, Y. (2014). Distinct Roles of Arrestin 1 in Photoreceptors During Drosophila Development. J Biol Chem [Epub ahead of print]. PubMed ID: 24838243
Summary: Arrestin regulates many facets of G-protein coupled receptors signaling. In Drosophila, Arrestin 1 (Arr1) is expressed at a lower level than Arrestin 2 (Arr2), and the role of Arr1 in visual physiology is less understood. This study generated transgenic flies expressing enhanced green fluorescent protein tagged Arr1 (Arr1-eGFP) and explored its trafficking in live photoreceptors. Arr1-eGFP is localized in the cytoplasm, and displays light-dependent translocation to the rhabdomere possibly by interacting with photoactivated Rh1 (Rh1*). In the adult, translocation of Arr1-eGFP occurs with slower kinetics when compared to that of Arr2-eGFP. This slower kinetics may be attributable to a reduced level of phosphorylated Rh1*. Indeed, a reduced level of phosphorylated Rh1* recruits a lower level of Arr1-eGFP to rhabdomeres. To investigate whether Arr1 is required for the deactivation of phosphorylated Rh1*, it was shown that in flies with reduced Arr1 prolonged depolarizing afterpotential (PDA) can be triggered with fewer light pulses, indicating that inactivation of phosphorylated Rh1* is compromised when the Arr1 level is reduced. Consistently, Arr1 is no longer required for deactivation of Rh1 in flies expressing phosphorylation deficient Rh1. Previously it was reported that Arr1 displays light-dependent internalization. Unexpectedly, in adult photoreceptors no endocytosis of Arr1-eGFP was observed. In contrast, it was shown that in pupal photoreceptors Arr1-eGFP becomes internalized and sequestered in vesicles within the cytoplasm. Taken together, it is proposed that Arr1 plays distinct roles during development and adulthood. Arr1 orchestrates the recycling of phosphorylated Rh1* in pupae while it regulates the deactivation in adult.

Meyer, S., Schmidt, I. and Klambt, C. (2014). Glia ECM interactions are required to shape the Drosophila nervous system. Mech Dev [Epub ahead of print]. PubMed ID: 24859129
Summary: Organs are characterized by a specific shape that is often remodeled during development. The dynamics of organ shape is in particular evident during the formation of the Drosophila nervous system. During embryonic stages the central nervous system compacts, whereas selective growth occurs during larval stages. The nervous system is covered by a layer of surface glial cells that form the blood brain barrier and a thick extracellular matrix called neural lamella. The size of the neural lamella is dynamically adjusted to the growing nervous system, and this study shows that perineurial glial cells secrete proteases to remodel this matrix. Moreover, an imbalance in proteolytic activity results in an abnormal shape of the nervous system. To identify further components controlling nervous system shape, an RNAi based screen was performed and the gene nolo, which encodes an ADAMTS-like protein, was identified. Loss of function alleles were generated, and a requirement in glial cells was demonstrated. Mutant nolo larvae, however, do not show an abnormal nervous system shape. The only predicted off-target of the nolodsRNA is Oatp30B, which encodes an organic anion transporting protein characterized by an extracellular protease inhibitor domain. Loss of function mutants were generated and double mutant analyses demonstrate a genetic interaction between nolo and Oatp30B which prevented the generation of maternal zygotic mutant larvae.

Hegde, V. R., Vogel, R. and Feany, M. B. (2014). Glia are critical for the neuropathology of complex I deficiency in Drosophila. Hum Mol Genet [Epub ahead of print]. PubMed ID: 24760769
Summary: Mitochondrial electron transport chain (ETC) disorders cause severe neurological disease, typically in the context of fatal encephalomyelopathies. Neuronal cell autonomous energy deficiency due to reduced mitochondrial ATP production is currently the leading hypothesis to explain the neurotoxicity in ETC disorders. To define the mechanisms underlying neuropathology in ETC disorders, this study modeled the most common type of ETC disorder, complex I deficiency, in Drosophila. The model recapitulates important clinical features of the disease including neuronal loss, mitochondrial enlargement, motor dysfunction and early death. Using cell-type specific gene knockdown, it was found that both neurons and glia contribute to the disease phenotype and that glia play a critical non-cell autonomous role in the development of neuronal toxicity. The results open up an unexpected avenue of research, and could lead to the development of new treatment strategies.

Tuesday, June 3rd

DasGupta, S., Ferreira, C. H. and Miesenbock, G. (2014). FoxP influences the speed and accuracy of a perceptual decision in Drosophila. Science 344: 901-904. PubMed ID: 24855268
Summary: Decisions take time if information gradually accumulates to a response threshold, but the neural mechanisms of integration and thresholding are unknown. This study characterized a decision process in Drosophila that bears the behavioral signature of evidence accumulation. As stimulus contrast in trained odor discriminations decreased, reaction times increased and perceptual accuracy declined, in quantitative agreement with a drift-diffusion model. FoxP mutants took longer than wild-type flies to form decisions of similar or reduced accuracy, especially in difficult, low-contrast tasks. RNA interference with FoxP expression in alphabeta core Kenyon cells, or the overexpression of a potassium conductance in these neurons, recapitulated the FoxP mutant phenotype. A mushroom body subdomain whose development or function require the transcription factor FoxP thus supports the progression of a decision toward commitment.

Tuthill, J. C., Nern, A., Rubin, G. M. and Reiser, M. B. (2014). Wide-field feedback neurons dynamically tune early visual processing. Neuron 82: 887-895. PubMed ID: 24853944
Summary: An important strategy for efficient neural coding is to match the range of cellular responses to the distribution of relevant input signals. However, the structure and relevance of sensory signals depend on behavioral state. This study shows that behavior modifies neural activity at the earliest stages of fly vision. A class of wide-field neurons is described that provides feedback to the most peripheral layer of the Drosophila visual system, the lamina. Using in vivo patch-clamp electrophysiology, it was found that lamina wide-field neurons respond to low-frequency luminance fluctuations. Recordings in flying flies revealed that the gain and frequency tuning of wide-field neurons change during flight, and that these effects are mimicked by the neuromodulator octopamine. Genetically silencing wide-field neurons increased behavioral responses to slow-motion stimuli. Together, these findings identify a cell type that is gated by behavior to enhance neural coding by subtracting low-frequency signals from the inputs to motion detection circuits.

Andrews, J. C., Fernandez, M. P., Yu, Q., Leary, G. P., Leung, A. K., Kavanaugh, M. P., Kravitz, E. A. and Certel, S. J. (2014). Octopamine neuromodulation regulates gr32a-linked aggression and courtship pathways in Drosophila males. PLoS Genet 10: e1004356. PubMed ID: 24852170
Summary: Chemosensory pheromonal information regulates aggression and reproduction in many species, but how pheromonal signals are transduced to reliably produce behavior is not well understood. This study demonstrates that the pheromonal signals detected by Gr32a-expressing chemosensory neurons to enhance male aggression are filtered through octopamine (OA, invertebrate equivalent of norepinephrine) neurons. Using behavioral assays, we find males lacking both octopamine and Gr32a gustatory receptors exhibit parallel delays in the onset of aggression and reductions in aggression. Physiological and anatomical experiments identify Gr32a to octopamine neuron synaptic and functional connections in the suboesophageal ganglion. Refining the Gr32a-expressing population indicates that mouth Gr32a neurons promote male aggression and form synaptic contacts with OA neurons. By restricting the monoamine neuron target population, it was shown that three previously identified OA-FruM neurons involved in behavioral choice are among the Gr32a-OA connections. These findings demonstrate that octopaminergic neuromodulatory neurons function as early as a second-order step in this chemosensory-driven male social behavior pathway.

Vasmer, D., Pooryasin, A., Riemensperger, T. and Fiala, A. (2014). Induction of aversive learning through thermogenetic activation of Kenyon cell ensembles in Drosophila. Front Behav Neurosci 8: 174. PubMed ID: 24860455
Summary: Drosophila represents a model organism to analyze neuronal mechanisms underlying learning and memory. Kenyon cells of the Drosophila mushroom body are required for associative odor learning and memory retrieval. But is the mushroom body sufficient to acquire and retrieve an associative memory? To answer this question an experimental approach was conceived to bypass olfactory sensory input and to thermogenetically activate sparse and random ensembles of Kenyon cells directly. It was found that if the artificial activation of Kenyon cell ensembles coincides with a salient, aversive stimulus learning was induced. The animals adjusted their behavior in a subsequent test situation and actively avoided reactivation of these Kenyon cells. These results show that Kenyon cell activity in coincidence with a salient aversive stimulus can suffice to form an associative memory. Memory retrieval is characterized by a closed feedback loop between a behavioral action and the reactivation of sparse ensembles of Kenyon cells.

Bath, D. E., Stowers, J. R., Hormann, D., Poehlmann, A., Dickson, B. J. and Straw, A. D. (2014). FlyMAD: rapid thermogenetic control of neuronal activity in freely walking Drosophila. Nat Methods [Epub ahead of print]. PubMed ID: 24859752
Summary: Rapidly and selectively modulating the activity of defined neurons in unrestrained animals is a powerful approach in investigating the circuit mechanisms that shape behavior. In Drosophila melanogaster, temperature-sensitive silencers and activators are widely used to control the activities of genetically defined neuronal cell types. A limitation of these thermogenetic approaches, however, has been their poor temporal resolution. This study introduced FlyMAD (the fly mind-altering device), which allows thermogenetic silencing or activation within seconds or even fractions of a second. Using computer vision, FlyMAD targets an infrared laser to freely walking flies. As a proof of principle, the rapid silencing and activation of neurons involved in locomotion, vision and courtship were demonstrated. The spatial resolution of the focused beam enabled preferential targeting of neurons in the brain or ventral nerve cord. Moreover, the high temporal resolution of FlyMAD allowed discovery of distinct timing relationships for two neuronal cell types previously linked to courtship song.

Monday, June 2nd

Bhaskar, P. K., Surabhi, S., Tripathi, B. K., Mukherjee, A. and Mutsuddi, M. (2014). dLin52 is crucial for dE2F and dRBF mediated transcriptional regulation of pro-apoptotic gene hid. Biochim Biophys Acta. PubMed ID: 24863159
Summary: Drosophila lin52 (dlin52) is a member of Myb transcription regulator complex and it shows a dynamic pattern of expression in all Drosophila tissues. Myb complex functions to activate or repress transcription in a site-specific manner; however, the detailed mechanism is yet to be clearly understood. Members of the Drosophila melanogaster Myb-MuvB/dREAM complex have been known to regulate expression of a wide range of genes including those involved in regulating apoptosis. E2F and its corepressor RBF also belong to this complex and together they regulate expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. The present study examined whether the depletion of dlin52 in developing photoreceptor neurons results in enhanced apoptosis and disorganisation of the ommatidia. Strikingly, dLin52 was found to be essential for transcriptional repression of the pro-apoptotic gene, hid; decrease in dlin52 levels led to dramatic induction of hid and apoptosis in eye-antennal discs. Reduction of Rpd3 (HDAC1), another member of the dREAM complex, also led to marginal upregulation of Hid. In addition, an optimum level of dLin52 was found to be needed for dE2F1/2 activity on the hid promoter. dlin52 cooperates with dRBF and dE2F1/2 for recruitment of repressor complex on the hid promoter. Preliminary data indicates that Rpd3/HDAC1 also contributes to hid repression. Based on the findings, it is concluded that dLin52 functions as a co-factor and modulates activity of members of dMyb/dREAM complex at hid promoter, thus, regulating apoptosis by repressing this pro-apoptotic gene in the developing Drosophila eye.

Sato-Miyata, Y., Muramatsu, K., Funakoshi, M., Tsuda, M. and Aigaki, T. (2014). Overexpression of dilp2 causes nutrient-dependent semi-lethality in Drosophila. Front Physiol 5: 147. PubMed ID: 24795642
Summary: Insulin/insulin-like growth factor (IGF) plays an important role as a systemic regulator of metabolism in multicellular organisms. Hyperinsulinemia, a high level of blood insulin, is often associated with impaired physiological conditions such as hypoglycemia, insulin resistance, and diabetes. However, due to the complex pathophysiology of hyperinsulinemia, the causative role of excess insulin/IGF signaling has remained elusive. To investigate the biological effects of a high level of insulin in metabolic homeostasis and physiology, flies were generated overexpressing Drosophila insulin-like peptide 2 (Dilp2), which has the highest potential of promoting tissue growth among the Ilp genes in Drosophila. In this model, a UAS-Dilp2 transgene was overexpressed under control of sd-Gal4 that drives expression predominantly in developing imaginal wing discs. Overexpression of Dilp2 caused semi-lethality, which was partially suppressed by mutations in the insulin receptor (InR) or Akt1, suggesting that dilp2-induced semi-lethality is mediated by the PI3K/Akt1 signaling. dilp2-overexpressing flies exhibited intensive autophagy in fat body cells. Interestingly, the dilp2-induced autophagy as well as the semi-lethality was partially rescued by increasing the protein content relative to glucose in the media. These results suggest that excess insulin/IGF signaling impairs the physiology of animals, which can be ameliorated by controlling the nutritional balance between proteins and carbohydrates, at least in flies.

Wang, X., Wang, Z., Chen, Y., Huang, X., Hu, Y., Zhang, R., Ho, M. S. and Xue, L. (2014). FoxO mediates APP-induced AICD-dependent cell death. Cell Death Dis 5: e1233. PubMed ID: 24832605
Summary: The amyloid precursor protein (APP) is a broadly expressed transmembrane protein that has a significant role in the pathogenesis of Alzheimer's disease (AD). APP can be cleaved at multiple sites to generate a series of fragments including the amyloid beta (Abeta) peptides and APP intracellular domain (AICD). Although Abeta peptides have been proposed to be the main cause of AD pathogenesis, the role of AICD has been underappreciated. This study reports that APP induces AICD-dependent cell death in Drosophila neuronal and non-neuronal tissues. A genetic screen identified the transcription factor forkhead box O (FoxO) as a crucial downstream mediator of APP-induced cell death and locomotion defect. In mammalian cells, AICD physically interacts with FoxO in the cytoplasm, translocates with FoxO into the nucleus upon oxidative stress, and promotes FoxO-induced transcription of pro-apoptotic gene Bim. These data demonstrate that APP modulates FoxO-mediated cell death through AICD, which acts as a transcriptional co-activator of FoxO.

Politi, Y., Gal, L., Kalifa, Y., Ravid, L., Elazar, Z. and Arama, E. (2014). Paternal mitochondrial destruction after fertilization is mediated by a common endocytic and autophagic pathway in Drosophila. Dev Cell 29: 305-320. PubMed ID: 24823375
Summary: Almost all animals contain mitochondria of maternal origin only, but the exact mechanisms underlying this phenomenon are still vague. This study investigated the fate of Drosophila paternal mitochondria after fertilization. The sperm mitochondrial derivative (MD) is rapidly eliminated in a stereotypical process dubbed paternal mitochondrial destruction (PMD). PMD is initiated by a network of vesicles resembling multivesicular bodies and displaying common features of the endocytic and autophagic pathways. These vesicles associate with the sperm tail and mediate the disintegration of its plasma membrane. Subsequently, the MD separates from the axoneme and breaks into smaller fragments, which are then sequestered by autophagosomes for degradation in lysosomes. Evidence is provided for the involvement of the ubiquitin pathway and the autophagy receptor p62 in this process. Finally, it was shown that the ubiquitin ligase Parkin is not involved in PMD, implying a divergence from the autophagic pathway of damaged mitochondria.

Lubkov, V. and Bar-Sagi, D. (2014). E-cadherin-mediated cell coupling is required for apoptotic cell extrusion. Curr Biol 24: 868-874. PubMed ID: 24704076
Summary: Apoptotic extrusion is a multicellular process utilized by live cells to remove neighboring apoptotic cells. In epithelial tissues, this process has been shown to be critical for the preservation of tissue integrity and barrier function. This study demonstrates that extrusion is driven by the retraction of the apoptotic cell, which, in turn, triggers a transient and coordinated elongation of the neighboring cells. The coordination of cell elongation requires E-cadherin-mediated cell-cell adhesion. Accordingly, cells that express low levels of E-cadherin (see Drosophila Shotgun) are compromised in elongation and apoptotic extrusion, and furthermore, display loss of barrier function in response to apoptotic stimuli. These findings indicate that the maintenance of adhesive forces during apoptotic cell turnover might play an essential role in controlling tissue homeostasis.

Sunday, June 1st

Rogulja-Ortmann, A., Picao-Osorio, J., Villava, C., Patraquim, P., Lafuente, E., Aspden, J., Thomsen, S., Technau, G. M. and Alonso, C. R. (2014). The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system. Development 141: 2046-2056. PubMed ID: 24803653
Summary: The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. These issues were explored within the developing Drosophila central nervous system (CNS). The pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches it was demonstrated that ELAV binds to discrete elements within Ubx RNAs and that their genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAV-dependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, it is noted that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes it is proposed that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation.

Guilgur, L. G., Prudencio, P., Sobral, D., Liszekova, D., Rosa, A. and Martinho, R. G. (2014). Requirement for highly efficient pre-mRNA splicing during Drosophila early embryonic development. Elife 3: e02181. PubMed ID: 24755291
Summary: Drosophila syncytial nuclear divisions limit transcription unit size of early zygotic genes. As mitosis inhibits not only transcription, but also pre-mRNA splicing, it was reasoned that constraints on splicing were likely to exist in the early embryo; splicing avoidance a possible explanation why most early zygotic genes are intronless. Two mutant alleles were isolated for a subunit of the NTC/Prp19 complex, Fandango, that specifically impaired pre-mRNA splicing of early zygotic but not maternally encoded transcripts. It was hypothesized that the requirements for pre-mRNA splicing efficiency were likely to vary during development. Ectopic maternal expression of an early zygotic pre-mRNA was sufficient to suppress its splicing defects in the mutant background. Furthermore, a small early zygotic transcript with multiple introns was poorly spliced in wild-type embryos. These findings demonstrate for the first time the existence of a developmental prerequisite for highly efficient splicing during Drosophila early embryonic development and suggest a need, in highly proliferative tissues, for coordination between cell cycle and gene architecture to ensure correct gene expression and avoid abnormally processed transcripts.

Williams, S. G. and Hall, K. B. (2014). Linkage and Allostery in snRNP Protein:RNA Complexes. Biochemistry [Epub ahead of print]. PubMed ID: 24849693
Summary: Drosophila SNF is a member of the U1A/U2B''/SNF protein family that is found in U1 and U2 snRNPs, where it binds to Stemloop II and Stemloop IV of U1 or U2 snRNA, respectively. In the U2 snRNP, SNF is also bound to the U2A' protein, which is not found in the U1 snRNP. Although previous reports have implicated U2A' as a necessary auxiliary protein for SNF binding to Stemloop IV, no thermodynamic analysis of the interactions has been done. Using in vitro RNA binding isotherms and isothermal titration calorimetry, the thermodynamics of SNF/RNA/U2A' ternary complex formation have been characterized. There is a very large cooperativity unique to Stemloop IV that explains the localization of U2A' to the U2 snRNP and the exclusion of U2A' from the U1 snRNP. The binding cooperativity, or heterotropic linkage, is interpreted with respect to linked conformational equilibria of both SNF and its RNA ligand. This represents an example of protein-RNA allostery.

Preussner, M., Wilhelmi, I., Schultz, A. S., Finkernagel, F., Michel, M., Moroy, T. and Heyd, F. (2014). Rhythmic U2af26 Alternative Splicing Controls PERIOD1 Stability and the Circadian Clock in Mice. Mol Cell 54: 651-662. PubMed ID: 24837677
Summary: The circadian clock drives daily rhythms in gene expression to control metabolism, behavior, and physiology; while the underlying transcriptional feedback loops are well defined, the impact of alternative splicing on circadian biology remains poorly understood. This paper describes a robust circadian and light-inducible splicing switch that changes the reading frame of the mouse mRNA encoding U2-auxiliary-factor 26 (U2AF26). This results in translation far into the 3' UTR, generating a C terminus with homology to the Drosophila clock regulator Timeless. This new U2AF26 variant destabilizes PERIOD1 protein (see Drosophila Period), and U2AF26-deficient mice show nearly arrhythmic PERIOD1 protein levels and broad defects in circadian mRNA expression in peripheral clocks. At the behavioral level, these mice display increased phase advance adaptation following experimental jet lag. These data suggest light-induced U2af26 alternative splicing to be a buffering mechanism that limits PERIOD1 induction, thus stabilizing the circadian clock against abnormal changes in light:dark conditions.

Algama, M., Oldmeadow, C., Tasker, E., Mengersen, K. and Keith, J. M. (2014). Drosophila 3' UTRs Are More Complex than Protein-Coding Sequences. PLoS One 9: e97336. PubMed ID: 24824035
Summary: The 3' UTRs of eukaryotic genes participate in a variety of post-transcriptional (and some transcriptional) regulatory interactions. Some of these interactions are well characterised, but an undetermined number remain to be discovered. While some regulatory sequences in 3' UTRs may be conserved over long evolutionary time scales, others may have only ephemeral functional significance as regulatory profiles respond to changing selective pressures. This paper proposes a sensitive segmentation methodology for investigating patterns of composition and conservation in 3' UTRs based on comparison of closely related species. Encodings are described of pairwise and three-way alignments integrating information about conservation, GC content and transition/transversion ratios, and the method is applied to three closely related Drosophila species: D. melanogaster, D. simulans and D. yakuba. Incorporating multiple data types greatly increased the number of segment classes identified compared to similar methods based on conservation or GC content alone. It is proposed that the number of segments and number of types of segments identified by the method can be used as proxies for functional complexity. The main finding is that the number of segments and segment classes identified in 3' UTRs is greater than in the same length of protein-coding sequence, suggesting greater functional complexity in 3' UTRs. There is thus a need for sustained and extensive efforts by bioinformaticians to delineate functional elements in this important genomic fraction.

Sunday, June 15th

Luo, J., Lushchak, O. V., Goergen, P., Williams, M. J. and Nassel, D. R. (2014). Drosophila insulin-producing cells are differentially modulated by serotonin and octopamine receptors and affect social behavior. PLoS One 9: e99732. PubMed ID: 24923784
Summary: A set of 14 insulin-producing cells (IPCs) in the Drosophila brain produces three insulin-like peptides (DILP2, 3 and 5). Activity in IPCs and release of DILPs is nutrient dependent and controlled by multiple factors such as fat body-derived proteins, neurotransmitters, and neuropeptides. Two monoamine receptors, the octopamine receptor OAMB and the serotonin receptor 5-HT1A, are expressed by the IPCs. These receptors may act antagonistically on adenylate cyclase. This study investigated the action of the two receptors on activity in and output from the IPCs. Knockdown of OAMB by targeted RNAi led to elevated Dilp3 transcript levels in the brain, whereas 5-HT1A knockdown resulted in increases of Dilp2 and 5. OAMB-RNAi in IPCs leads to extended survival of starved flies and increased food intake, whereas 5-HT1A-RNAi produces the opposite phenotypes. However, knockdown of either OAMB or 5-HT1A in IPCs both lead to increased resistance to oxidative stress. In assays of carbohydrate levels it was found that 5-HT1A knockdown in IPCs resulted in elevated hemolymph glucose, body glycogen and body trehalose levels, while no effects were seen after OAMB knockdown. It was also found that manipulations of the two receptors in IPCs affected male aggressive behavior in different ways and 5-HT1A-RNAi reduced courtship latency. These observations suggest that activation of 5-HT1A and OAMB signaling in IPCs generates differential effects on Dilp transcription, fly physiology, metabolism and social interactions. However the findings do not support an antagonistic action of the two monoamines and their receptors in this particular system.

Pineiro, C., Lopes, C. S. and Casares, F. (2014). A conserved transcriptional network regulates lamina development in the Drosophila visual system. Development [Epub ahead of print]. PubMed ID: 24924198
Summary: The visual system of insects is a multilayered structure composed externally by the compound eye and internally by the three ganglia of the optic lobe: lamina, medulla and the lobula complex. The differentiation of lamina neurons depends heavily on Hedgehog (Hh) signaling, which is delivered by the incoming photoreceptor axons, and occurs in a wave-like fashion. Despite the primary role of lamina neurons in visual perception, it is still unclear how these neurons are specified from neuroepithelial (NE) progenitors. This study shows that a homothorax (hth) - eyes absent (eya) - sine oculis (so) - dachshund (dac) gene regulatory cassette is involved in this specification. Lamina neurons differentiate from NE progenitors that express hth, eya and so. One of the first events in the differentiation of lamina neurons is the upregulation of dac expression in response to Hh signaling. This dac upregulation, which marks the transition from NE progenitors into lamina precursors, also requires Eya/So, the expression of which is locked in by mutual feedback. dac expression is crucial for lamina differentiation because it ensures repression of hth, a negative regulator of single-minded, and thus dac allows further lamina neuron differentiation. Therefore, the specification of lamina neurons is controlled by coupling the cell-autonomous hth-eya-so-dac regulatory cassette to Hh signaling.

Jiang, N., Soba, P., Parker, E., Kim, C. C. and Parrish, J. Z. (2014). The microRNA bantam regulates a developmental transition in epithelial cells that restricts sensory dendrite growth. Development [Epub ahead of print]. PubMed ID: 24924190
Summary: As animals grow, many early born structures grow by cell expansion rather than cell addition; thus growth of distinct structures must be coordinated to maintain proportionality. This phenomenon is particularly widespread in the nervous system, with dendrite arbors of many neurons expanding in concert with their substrate to sustain connectivity and maintain receptive field coverage as animals grow. After rapidly growing to establish body wall coverage, dendrites of Drosophila class IV dendrite arborization (C4da) neurons grow synchronously with their substrate, the body wall epithelium, providing a system to study how proportionality is maintained during animal growth. This study shows that the microRNA bantam (ban) ensures coordinated growth of C4da dendrites and the epithelium through regulation of epithelial endoreplication, a modified cell cycle that entails genome amplification without cell division. In Drosophila larvae, epithelial endoreplication leads to progressive changes in dendrite-extracellular matrix (ECM) and dendrite-epithelium contacts, coupling dendrite/substrate expansion and restricting dendrite growth beyond established boundaries. Moreover, changes in epithelial expression of cell adhesion molecules, including the beta-integrin Myospheroid (Mys), accompany this developmental transition. Finally, endoreplication and the accompanying changes in epithelial mys expression are required to constrain late-stage dendrite growth and structural plasticity. Hence, modulating epithelium-ECM attachment probably influences substrate permissivity for dendrite growth and contributes to the dendrite-substrate coupling that ensures proportional expansion of the two cell types.

Okigawa, S., Mizoguchi, T., Okano, M., Tanaka, H., Isoda, M., Jiang, Y. J., Suster, M., Higashijima, S., Kawakami, K. and Itoh, M. (2014). Different combinations of Notch ligands and receptors regulate V2 interneuron progenitor proliferation and V2a/V2b cell fate determination. Dev Biol 391: 196-206. PubMed ID: 24768892
Summary: The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Using zebrafish embryos with altered Notch signaling, this study shows that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD (see Drosophila Delta), and three receptors, Notch1a, Notch1b, and Notch3 (see Drosophila Notch) redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib (see Drosophila Mind bomb), which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand-receptor combinations that occur concurrently during development in zebrafish.

Saturday, June 14th

Scopelliti, A., Cordero, J. B., Diao, F., Strathdee, K., White, B. H., Sansom, O. J. and Vidal, M. (2014). Local Control of Intestinal Stem Cell Homeostasis by Enteroendocrine Cells in the Adult Drosophila Midgut. Curr Biol 24(11): 1199-211. PubMed ID: 24814146
Summary: Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream. This paper reports a novel function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the Drosophila midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts - beyond its known roles in development - as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2 (Rickets), the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP. This study therefore has identified a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells.

Huo, Y. and Macara, I. G. (2014). The Par3-like polarity protein Par3L is essential for mammary stem cell maintenance. Nat Cell Biol 16: 529-537. PubMed ID: 24859006
Summary: The Par polarity proteins play key roles in asymmetric division of Drosophila melanogaster stem cells; however, whether the same mechanisms control stem cells in mammals is controversial. Although necessary for mammary gland morphogenesis, Par3 (Drosophila homolog: Bazooka) is not essential for mammary stem cell function. This study discovered that, instead, a previously uncharacterized protein, Par3-like (Par3L), is vital for mammary gland stem cell maintenance. Par3L function has been mysterious because, unlike Par3, it does not interact with atypical protein kinase C or the Par6 polarity protein. Par3L was found to be expressed by multipotent stem cells in the terminal end buds of murine mammary glands. Ablation of Par3L resulted in rapid and profound stem cell loss. Unexpectedly, Par3L, but not Par3, binds to the tumour suppressor protein Lkb1 (see Drosophila Lkb1) and inhibits its kinase activity. This interaction is key for the function of Par3L in mammary stem cell maintenance. These data reveal insights into a link between cell polarity proteins and stem cell survival, and uncover a biological function for Par3L.

Rogers, R. P. and Rogina, B. (2014). Increased mitochondrial biogenesis preserves intestinal stem cell homeostasis and contributes to longevity in Indy mutant flies. Aging (Albany NY) 6: 335-350. PubMed ID: 24827528
Summary: The Drosophila Indy (I'm not dead yet) gene encodes a plasma membrane transporter of Krebs cycle intermediates, with robust expression in tissues associated with metabolism. Reduced INDY alters metabolism and extends longevity in a manner similar to caloric restriction (CR); however, little is known about the tissue specific physiological effects of INDY reduction. This study focused on the effects of INDY reduction in the Drosophila midgut due to the importance of intestinal tissue homeostasis in healthy aging and longevity. The expression of Indy mRNA in the midgut changes in response to aging and nutrition. Genetic reduction of Indy expression increases midgut expression of the mitochondrial regulator spargel/dPGC-1, which is accompanied by increased mitochondrial biogenesis and reduced reactive oxygen species (ROS). These physiological changes in the Indy mutant midgut preserve intestinal stem cell (ISC) homeostasis and are associated with healthy aging. Genetic studies confirm that dPGC-1 mediates the regulatory effects of INDY, as illustrated by lack of longevity extension and ISC homeostasis in flies with mutations in both Indy and dPGC1. These data suggest INDY may be a physiological regulator that modulates intermediary metabolism in response to changes in nutrient availability and organismal needs by modulating dPGC-1.

Vogg, M. C., Owlarn, S., Perez Rico, Y. A., Xie, J., Suzuki, Y., Gentile, L., Wu, W. and Bartscherer, K. (2014). Stem cell-dependent formation of a functional anterior regeneration pole in planarians requires Zic and Forkhead transcription factors. Dev Biol 390: 136-148. PubMed ID: 24704339
Summary: Planarians can regenerate their head within days. This process depends on the direction of adult stem cells to wound sites and the orchestration of their progenitors to commit to appropriate lineages and to arrange into patterned tissues. This study identified a zinc finger transcription factor, Smed-ZicA (see Drosophila Odd Paired)), as a downstream target of Smed-FoxD (see Drosophila Fork head), a Forkhead transcription factor required for head regeneration. Smed-zicA and Smed-FoxD are co-expressed with the Wnt inhibitor notum and the Activin inhibitor follistatin in a cluster of cells at the anterior-most tip of the regenerating head - the anterior regeneration pole - and in surrounding stem cell progeny. Depletion of Smed-zicA and Smed-FoxD by RNAi abolishes notum and follistatin expression at the pole and inhibits head formation downstream of initial polarity decisions. A model is suggested in which ZicA and FoxD transcription factors synergize to control the formation of Notum- and Follistatin-producing anterior pole cells. Pole formation might constitute an early step in regeneration, resulting in a signaling center that orchestrates cellular events in the growing tissue.

Friday, June 13

Lin, Y. H., Chen, Y. C., Kao, T. Y., Lin, Y. C., Hsu, T. E., Wu, Y. C., Ja, W. W., Brummel, T. J., Kapahi, P., Yuh, C. H., Yu, L. K., Lin, Z. H., You, R. J., Jhong, Y. T. and Wang, H. D. (2014). Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans. Aging Cell [Epub ahead of print]. PubMed ID: 24889782
Summary: Target of rapamycin (TOR) signaling is a nutrient-sensing pathway controlling metabolism and lifespan. Although TOR signaling can be activated by a metabolite of diacylglycerol (DAG), phosphatidic acid (PA), the precise genetic mechanism through which DAG metabolism influences lifespan remains unknown. DAG is metabolized to either PA via the action of DAG kinase or 2-arachidonoyl-sn-glycerol by diacylglycerol lipase (DAGL). This study reports that in Drosophila and Caenorhabditis elegans, overexpression of diacylglycerol lipase (DAGL/inaE/dagl-1) or knockdown of diacylglycerol kinase (DGK/rdgA/dgk-5) extends lifespan and enhances response to oxidative stress. Phosphorylated S6 kinase (p-S6K) levels are reduced following these manipulations, implying the involvement of TOR signaling. Conversely, DAGL/inaE/dagl-1 mutants exhibit shortened lifespan, reduced tolerance to oxidative stress, and elevated levels of p-S6K. Additional results from genetic interaction studies are consistent with the hypothesis that DAG metabolism interacts with TOR and S6K signaling to affect longevity and oxidative stress resistance. These findings highlight conserved metabolic and genetic pathways that regulate aging.

Kon, N., Yoshikawa, T., Honma, S., Yamagata, Y., Yoshitane, H., Shimizu, K., Sugiyama, Y., Hara, C., Kameshita, I., Honma, K. and Fukada, Y. (2014). CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms. Genes Dev 28: 1101-1110. PubMed ID: 24831701
Summary: Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca(2+) in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. This study shows that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII; see Drosophila CamKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIalpha weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca(2+)-calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of CLOCK and BMAL1 (see Drosophila Clock and Cycle). These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities.

Fantauzzo, K. A. and Soriano, P. (2014). PI3K-mediated PDGFRalpha signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways. Genes Dev 28: 1005-1017. PubMed ID: 24788519
Summary: Previous studies have identified phosphatidylinositol 3-kinase (PI3K; see Drosophila Pi3K92E) as the main downstream effector of PDGFRalpha signaling during murine skeletal development. Autophosphorylation mutant knock-in embryos in which PDGFRalpha (See Drosophila Pvr) is unable to bind PI3K (Pdgfra(PI3K/PI3K)) exhibit skeletal defects affecting the palatal shelves, shoulder girdle, vertebrae, and sternum. To identify proteins phosphorylated by Akt (see Drosophila Akt1) downstream from PI3K-mediated PDGFRalpha signaling, Akt phosphorylation substrates from PDGF-AA-treated primary mouse embryonic palatal mesenchyme (MEPM) lysates were immunoprecipitated, and the peptides were analyzed by nanoliquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). This analysis generated a list of 56 proteins, including 10 that regulate cell survival and proliferation. It was demonstrated that MEPM cell survival is impaired in the presence of a PI3K inhibitor and that Pdgfra(PI3K/PI3K)-derived MEPMs do not proliferate in response to PDGF-AA treatment. Several of the identified Akt phosphorylation targets, including Ybox1, mediate cell survival through regulation of p53 (see Drosophila p53). Ybox1 binds both the p53 promoter and the p53 protein and expression of p53 is significantly decreased upon PDGF-AA treatment in MEPMs. Finally, this study demonstrated that introduction of a p53-null allele attenuates the vertebral defects found in Pdgfra(PI3K/PI3K) neonates. These findings identify p53 as a novel effector downstream from PI3K-engaged PDGFRalpha signaling that regulates survival and proliferation during skeletal development in vivo.

Bhandari, D., Raisch, T., Weichenrieder, O., Jonas, S. and Izaurralde, E. (2014). Structural basis for the Nanos-mediated recruitment of the CCR4-NOT complex and translational repression. Genes Dev 28: 888-901. PubMed ID: 24736845
Summary: The RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4-NOT deadenylase complex. This study shows that the three human Nanos paralogs (Nanos1-3) interact with the CNOT1 (see Drosophila Not1) C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1-3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1-3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. These findings provide the structural basis for the recruitment of the CCR4-NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4-NOT complex as the main effector complex for Nanos function.

Thursday, June 12th

Schwabe, T., Borycz, J. A., Meinertzhagen, I. A. and Clandinin, T. R. (2014). Differential Adhesion Determines the Organization of Synaptic Fascicles in the Drosophila Visual System. Curr Biol [Epub ahead of print]. PubMed ID: 24881879
Summary: Neuronal circuits in worms, flies, and mammals are organized so as to minimize wiring length for a functional number of synaptic connections, a phenomenon called wiring optimization. However, the molecular mechanisms that establish optimal wiring during development are unknown. This question was addressed by studying the role of N-cadherin in the development of optimally wired neurite fascicles in the peripheral visual system of Drosophila. Photoreceptor axons surround the dendrites of their postsynaptic targets, called lamina cells, within a concentric fascicle called a cartridge. N-cadherin is expressed at higher levels in lamina cells than in photoreceptors, and all genetic manipulations that invert these relative differences displace lamina cells to the periphery and relocate photoreceptor axon terminals into the center. It is concluded that Differential expression of a single cadherin is both necessary and sufficient to determine cartridge structure because it positions the most-adhesive elements that make the most synapses at the core and the less-adhesive elements that make fewer synapses at the periphery. These results suggest a general model by which differential adhesion can be utilized to determine the relative positions of axons and dendrites to establish optimal wiring.

Tanaka-Matakatsu, M., Miller, J., Borger, D., Tang, W. J. and Du, W. (2014). Daughterless homodimer synergizes with Eyeless to induce atonal expression and retinal neuron differentiation. Dev Biol [Epub ahead of print]. PubMed ID: 24886829
Summary: Class I Basic Helix-Loop-Helix (bHLH) transcription factors form homodimers or heterodimers with class II bHLH proteins. While bHLH heterodimers are known to have diverse roles, little is known about the role of class I homodimers. This study shows that a linked dimer of Daughterless (Da), the only Drosophila class I bHLH protein, activates Atonal (Ato) expression and retinal neuron differentiation synergistically with the retinal determination factor Eyeless (Ey). The HLH protein Extramacrocheate (Emc), which forms heterodimer with Da, antagonizes the synergistic activation from Da but not the Da-Da linked dimer with Ey. Da directly interacts with Ey and promotes Ey binding to the Ey binding site in the Ato 3' enhancer. Interestingly, the Ey binding site in the Ato 3' enhancer contains an embedded E-box that is also required for the synergistic activation by Ey and Da. Finally it was shown that mammalian homologs of Ey and Da can functionally replace their Drosophila counterparts to synergistically activate the Ato enhancer, suggesting that the observed function is evolutionary conserved.

Podufall, J., Tian, R., Knoche, E., Puchkov, D., Walter, A. M., Rosa, S., Quentin, C., Vukoja, A., Jung, N., Lampe, A., Wichmann, C., Bohme, M., Depner, H., Zhang, Y. Q., Schmoranzer, J., Sigrist, S. J. and Haucke, V. (2014). Presynaptic Role for the Cytomatrix Protein GIT in Synaptic Vesicle Recycling. Cell Rep [Epub ahead of print]. PubMed ID: 24882013
Summary: Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. This study identified the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and Stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.

Fernandes, V. M., Panchapakesan, S. S., Braid, L. R. and Verheyen, E. M. (2014). Nemo promotes Notch-mediated lateral inhibition downstream of proneural factors. Dev Biol [Epub ahead of print]. PubMed ID: 24880113
Summary: During neurogenesis, conserved tissue-specific proneural factors establish a cells competence to take on neural fate from within a field of unspecified cells. Proneural genes encode basic helix-loop-helix transcription factors that promote the expression of 'core' and subtype-specific target genes. Target genes include both pan-neuronal genes and genes that aid in the process of refinement, known as lateral inhibition. In this process, proneural gene expression is increased in the neural progenitor while simultaneously down-regulated in the surrounding cells, in a Notch signalling-dependent manner. This study identified nemo (nmo) as a target of members of both Drosophila Atonal and Achaete-Scute proneural factor families and found that mammalian proneural homologs induce Nemo-like-kinase (Nlk) expression in cell culture. nmo loss of function was found to lead to reduced expression of Notch targets and to perturbations in Notch-mediated lateral inhibition. Furthermore, Notch hyperactivity can compensate for nmo loss in the Drosophila eye. Thus nmo promotes Notch-mediated lateral inhibition downstream of proneural factors during neurogenesis.

Wednesday, June 11th

Wang, Z., Fan, M., Candas, D., Zhang, T. Q., Qin, L., Eldridge, A., Wachsmann-Hogiu, S., Ahmed, K. M., Chromy, B. A., Nantajit, D., Duru, N., He, F., Chen, M., Finkel, T., Weinstein, L. S. and Li, J. J. (2014). Cyclin B1/Cdk1 coordinates mitochondrial respiration for cell-cycle G2/M progression. Dev Cell 29: 217-232. PubMed ID: 24746669
Summary: A substantial amount of mitochondrial energy is required for cell-cycle progression. The mechanisms underlying the coordination of the mitochondrial respiration with cell-cycle progression, especially the G2/M transition, remain to be elucidated. This study shows that a fraction of cyclin B1/Cdk1 proteins (see Drosophila Cyclin B and Cdc2) localizes to the matrix of mitochondria and phosphorylates a cluster of mitochondrial proteins, including the complex I (CI) subunits in the respiratory chain. Cyclin B1/Cdk1-mediated CI phosphorylation enhances CI activity, whereas deficiency of such phosphorylation in each of the relevant CI subunits results in impairment of CI function. Mitochondria-targeted cyclin B1/Cdk1 increases mitochondrial respiration with enhanced oxygen consumption and ATP generation, which provides cells with efficient bioenergy for G2/M transition and shortens overall cell-cycle time. Thus, cyclin B1/Cdk1-mediated phosphorylation of mitochondrial substrates allows cells to sense and respond to increased energy demand for G2/M transition and, subsequently, to upregulate mitochondrial respiration for successful cell-cycle progression.

Li, D., Sakuma, R., Vakili, N. A., Mo, R., Puviindran, V., Deimling, S., Zhang, X., Hopyan, S. and Hui, C. C. (2014). Formation of proximal and anterior limb skeleton requires early function of irx3 and irx5 and is negatively regulated by shh signaling. Dev Cell 29: 233-240. PubMed ID: 24726282
Summary: Limb skeletal pattern relies heavily on graded Sonic hedgehog (Shh) signaling. As a morphogen and growth cue, Shh regulates identities of posterior limb elements, including the ulna/fibula and digits 2 through 5. In contrast, proximal and anterior structures, including the humerus/femur, radius/tibia, and digit 1, are regarded as Shh independent, and mechanisms governing their specification are unclear. This study shows that patterning of the proximal and anterior limb skeleton involves two phases. Irx3 and Irx5 (Irx3/5; see Drosophila Mirror) are essential in the initiating limb bud to specify progenitors of the femur, tibia, and digit 1. However, these skeletal elements can be restored in Irx3/5 null mice when Shh signaling is diminished, indicating that Shh negatively regulates their formation after initiation. These data provide genetic evidence supporting the concept of early specification and progressive determination of anterior limb pattern.

Zhulyn, O., Li, D., Deimling, S., Vakili, N. A., Mo, R., Puviindran, V., Chen, M. H., Chuang, P. T., Hopyan, S. and Hui, C. C. (2014). A switch from low to high shh activity regulates establishment of limb progenitors and signaling centers. Dev Cell 29: 241-249. PubMed ID: 24726283
Summary: The patterning and growth of the embryonic vertebrate limb is dependent on Sonic hedgehog (Shh; see Drosophila Hedgehog), a morphogen that regulates the activity of Gli transcription factors (see Drosophila Cubitus interuptus). However, Shh expression is not observed during the first 12 hr of limb development. During this phase, the limb bud is prepatterned into anterior and posterior regions through the antagonistic actions of transcription factors Gli3 and Hand2 (see Drosophila Hand). This study demonstrates that precocious activation of Shh signaling during this early phase interferes with the Gli3-dependent specification of anterior progenitors, disturbing establishment of signaling centers and normal outgrowth of the limb. These findings illustrate that limb development requires a sweet spot in the level and timing of pathway activation that allows for the Shh-dependent expansion of posterior progenitors without interfering with early prepatterning functions of Gli3/Gli3R or specification of anterior progenitors.

Smykal, V., Daimon, T., Kayukawa, T., Takaki, K., Shinoda, T. and Jindra, M. (2014). Importance of juvenile hormone signaling arises with competence of insect larvae to metamorphose. Dev Biol 390: 221-230. PubMed ID: 24662045
Summary: Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori, were chosed. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH, were examined. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, it is proposed that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth.

Tuesday, June 10th

Petkova, M. D., Little, S. C., Liu, F. and Gregor, T. (2014). Maternal Origins of Developmental Reproducibility. Curr Biol [Epub ahead of print]. PubMed ID: 24856210
Summary: Cell fate decisions during multicellular development are precisely coordinated, leading to highly reproducible macroscopic structural outcomes. The origins of this reproducibility are found at the molecular level during the earliest stages of development when patterns of morphogen molecules emerge reproducibly. However, although the initial conditions for these early stages are determined by the female during oogenesis, it is unknown whether reproducibility is perpetuated from oogenesis or reacquired by the zygote. To address this issue in the early Drosophila embryo, this study sought to count individual maternally deposited bicoid mRNA molecules and compare variability between embryos with previously observed fluctuations in the Bicoid protein gradient. Independent methods were developed to quantify total amounts of mRNA in individual embryos and show that mRNA counts are highly reproducible between embryos to within approximately 9%, matching the reproducibility of the protein gradient. Reproducibility emerges from perfectly linear feedforward processes: changing the genetic dosage in the female leads to proportional changes in both mRNA and protein numbers in the embryo. These results indicate that the reproducibility of the morphological structures of embryos originates during oogenesis, which is when the expression of maternally provided patterning factors is precisely controlled.

Liu, W. J., Reece-Hoyes, J. S., Walhout, A. J. and Eisenmann, D. M. (2014). Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6. BMC Dev Biol 14: 17. PubMed ID: 24885717
Summary: Hox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 (homolog of Drosophila Sex-combs reduced) is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, transcription factors necessary for lin-39 expression in the VPCs, factors were sought that initiate lin-39 expression in the embryo. The yeast one-hybrid (Y1H) method was used to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and several factors were characterized by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations inlin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site that was identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs. These results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintains lin-39 expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression in C. elegans is complicated, redundant and robust.

Kronja, I., Yuan, B., Eichhorn, S. W., Dzeyk, K., Krijgsveld, J., Bartel, D. P. and Orr-Weaver, T. L. (2014). Widespread Changes in the Posttranscriptional Landscape at the Drosophila Oocyte-to-Embryo Transition. Cell Rep [Epub ahead of print]. PubMed ID: 24882012
Summary: The oocyte-to-embryo transition marks the onset of development. The initial phase of this profound change from the differentiated oocyte to the totipotent embryo occurs in the absence of both transcription and mRNA degradation. This study combined global polysome profiling, ribosome-footprint profiling, and quantitative mass spectrometry in a comprehensive approach to delineate the translational and proteomic changes that occur during this important transition in Drosophila. The results show that Pan gu (PNG) kinase is a critical regulator of the extensive changes in the translatome, acting uniquely at this developmental window. Analysis of the proteome in png) mutants provided insights into the contributions of translation to changes in protein levels, revealing a compensatory dynamic between translation and protein turnover during proteome remodeling at the return to totipotency. The proteome changes additionally suggested regulators of meiosis and early embryogenesis, including the conserved H3K4 demethylase LID, which was demonstrated to be required during this period despite transcriptional inactivity.

Wisotzkey, R. G., Quijano, J. C., Stinchfield, M. J. and Newfeld, S. J. (2014). New gene evolution in the Bonus-TIF1-gamma/TRIM33 family impacted the architecture of the vertebrate dorsal-ventral patterning network. Mol Biol Evol. PubMed ID: 24881051
Summary: Uncovering how a new gene acquires its function and understanding how the function of a new gene influences existing genetic networks are important topics in evolutionary biology. This study demonstrated nonconservation for the embryonic functions of Drosophila Bonus and its newest vertebrate relative TIF1-gamma/TRIM33. It was shown previously that TIF1-gamma/TRIM33 functions as an ubiquitin ligase for the Smad4 (see Drosophila Medea) signal transducer and antagonizes the Bone Morphogenetic Protein (BMP) signaling network underlying vertebrate dorsal-ventral axis formation. This study showed that Bonus functions as an agonist of the Decapentaplegic (Dpp) signaling network underlying dorsal-ventral axis formation in flies. The absence of conservation for the roles of Bonus and TIF1-gamma/TRIM33 reveals a shift in the dorsal-ventral patterning networks of flies and mice, systems that were previously considered wholly conserved. The shift occurred when the new gene TIF1-gamma/TRIM33 replaced the function of the ubiquitin ligase Nedd4L in the lineage leading to vertebrates. Evidence of this replacement is the demonstration that Nedd4 performs the function of TIF1-gamma/TRIM33 in flies during dorsal-ventral axis formation. The replacement allowed vertebrate Nedd4L to acquire novel functions as a ubiquitin ligase of vertebrate-specific Smad proteins. Overall this data reveals that the architecture of the Dpp/BMP dorsal-ventral patterning network continued to evolve in the vertebrate lineage, after separation from flies, via the incorporation of new genes.

Monday, June 9th

Ostojic, I., Boll, W., Waterson, M. J., Chan, T., Chandra, R., Pletcher, S. D. and Alcedo, J. (2014). Positive and negative gustatory inputs affect Drosophila lifespan partly in parallel to dFOXO signaling. Proc Natl Acad Sci U S A. PubMed ID: 24847072
Summary: In Caenorhabditis elegans, a subset of gustatory neurons, as well as olfactory neurons, shortens lifespan, whereas a different subset of gustatory neurons lengthens it. Recently, the lifespan-shortening effect of olfactory neurons has been reported to be conserved in Drosophila. The Drosophila gustatory system also affects lifespan in a bidirectional manner. Taste inputs were found to shorten lifespan through inhibition of the insulin pathway effector dFOXO, whereas other taste inputs lengthen lifespan in parallel to this pathway. It was also noted that the gustatory influence on lifespan does not necessarily depend on food intake levels. Finally, the nature of some of the taste inputs that could shorten versus lengthen lifespan was identified. Together these data suggest that different gustatory cues can modulate the activities of distinct signaling pathways, including different insulin-like peptides, to promote physiological changes that ultimately affect lifespan.

Yu, J., Chia, J., Canning, C. A., Jones, C. M., Bard, F. A. and Virshup, D. M. (2014). WLS Retrograde Transport to the Endoplasmic Reticulum during Wnt Secretion. Dev Cell 29: 277-291. PubMed ID: 24768165
Summary: Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. This study found that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. An ER-targeting sequence was identified at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle.

Yau, R. G., Peng, Y., Valiathan, R. R., Birkeland, S. R., Wilson, T. E. and Weisman, L. S. (2014). Release from myosin V via regulated recruitment of an E3 ubiquitin ligase controls organelle localization. Dev Cell 28: 520-533. PubMed ID: 24636257
Summary: Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin V, Myo2 (see Drosophila Didum), binds the vacuole-specific adaptor Vac17 to transport the vacuole from the mother cell to the bud. This study shows that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 that recruits the E3 ligase Dma1 to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, it was found that the termination of peroxisome transport also requires Dma1. It is predicted that this is a general mechanism that detaches myosin V from select cargoes.

Mahesh, G., Jeong, E., Ng, F. S., Liu, Y., Gunawardhana, K., Houl, J. H., Yildirim, E., Amunugama, R., Jones, R., Allen, D. L., Edery, I., Kim, E. Y. and Hardin, P. E. (2014). Phosphorylation of the Transcription Activator CLOCK Regulates Progression Through a ~24h Feedback Loop to Influence Circadian Period in Drosophila. J Biol Chem [Epub ahead of print]. PubMed ID: 24872414
Summary: Circadian (~24hr) clocks control daily rhythms in metabolism, physiology and behavior in animals, plants and microbes. In Drosophila, these clocks keep circadian time via transcriptional feedback loops in which Clock-Cycle (Clk-Cyc) initiates transcription of period (per) and timeless (tim), accumulating levels of Per and Tim feed back to inhibit Clk-Cyc, and degradation of Per and Tim allows Clk-Cyc to initate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in Per and Clk phosphorylation, where Per and Clk phosphorylation is high during transcriptional repression. Per phosphorylation at specific sites controls its subcellular localization, activity and stability, but comparatively little is known about the identity and function of Clk phosphorylation sites. This study identified eight Clk phosphorylation sites via mass spectrometry and determined how phosphorylation at these sites impacts behavioral and molecular rhythms by transgenic rescue of a new Clk null mutant. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten circadian period, whereas loss of Clk phosphorylation at serine 859 increases Clk activity, thereby increasing Per levels and accelerating transcriptional repression. These results demonstrate that Clk phosphorylation influences circadian period by regulating Clk activity and progression through the feedback loop.

Sunday, June 8th

Cordero, J. B., Ridgway, R. A., Valeri, N., Nixon, C., Frame, M. C., Muller, W. J., Vidal, M. and Sansom, O. J. (2014). c-Src drives intestinal regeneration and transformation. EMBO J [Epub ahead of print]. PubMed ID: 24788409
Summary: The non-receptor tyrosine kinase c-Src, hereafter referred to as Src, is overexpressed or activated in multiple human malignancies. There has been much speculation about the functional role of Src in colorectal cancer (CRC), with Src amplification and potential activating mutations in up to 20% of the human tumours, although this has never been addressed due to multiple redundant family members. This study used the adult Drosophila and mouse intestinal epithelium as paradigms to define a role for Src during tissue homeostasis, damage-induced regeneration and hyperplasia. Through genetic gain and loss of function experiments, it was demonstrated that Src is necessary and sufficient to drive intestinal stem cell (ISC) proliferation during tissue self-renewal, regeneration and tumourigenesis. Surprisingly, Src plays a non-redundant role in the mouse intestine, which cannot be substituted by the other family kinases Fyn and Yes. Mechanistically, it was shown that Src drives ISC proliferation through upregulation of EGFR and activation of Ras/MAPK and Stat3 signalling. Therefore, this study demonstrated a novel essential role for Src in intestinal stem/progenitor cell proliferation and tumourigenesis initiation in vivo.

Geng, J., Xia, L., Li, W. and Dou, F. (2014). The C-Terminus of Tau protein plays an important role in its stability and toxicity. J Mol Neurosci [Epub ahead of print]. PubMed ID: 24788226
Summary: The identification of tau fragments generated by proteolysis in the neurons of AD patients and in neurofibrillary tangles encourages research on the toxicity of truncated tau. However, the detailed mechanism underlying the proteolysis-induced toxicity of tau is not clear and even controversial in some cases. The study used Drosophila as a model to evaluate the toxicity of a set of truncated tau fragments in vivo and found that the flies harboring C-terminal-truncated tau exhibited less toxicity due to the unstable characteristic of C-terminal-truncated tau fragments. Further study carried out in cultured Drosophila Kc cells revealed that C-terminal-truncated tau fragments degrade faster than full-length tau or N-terminal-truncated fragments. Collectively, these data implicate proteolysis of tau as an important pathway mediating tau degradation and neurotoxicity in vivo.

Schutte, R. J., Schutte, S. S., Algara, J., Barragan, E. V., Gilligan, J., Staber, C., Savva, Y. A., Smith, M. A., Reenan, R. and O'Dowd, D. K. (2014). Knock-in model of Dravet Syndrome reveals a constitutive and conditional reduction in sodium current. J Neurophysiol [Epub ahead of print]. PubMed ID: 24805083
Summary: Hundreds of mutations in the SCN1A sodium channel gene confer a wide spectrum of epileptic disorders, requiring efficient model systems to study cellular mechanisms and identify potential therapeutic targets. It has been demonstrated that Drosophila knock-in flies carrying a Generalized epilepsy with febrile seizures plus (GEFS+) causing SCN1A mutation (K1270T) results in a primarily conditional increase in sodium current activity that contributes to a heat-induced seizure phenotype. To determine whether different SCN1A mutations cause distinct phenotypes in Drosophila as they do in humans, this paper focuses on a knock-in line carrying a mutation that causes a more severe seizure disorder, Dravet Syndrome (DS), in humans. Introduction of the DS SCN1A mutation (S1231R) into the Drosophila sodium channel gene, para, results in flies that exhibit spontaneous and heat-induced seizures with distinct characteristics and lower onset temperature than the GEFS+ flies. Electrophysiological studies of GABAergic interneurons in the brains of adult DS flies reveal, for the first time in an in vivo model system, that a missense DS mutation causes a constitutive and conditional reduction in sodium current activity and repetitive firing. In addition, feeding with the serotonin precursor 5-HTP suppresses heat-induced seizures in DS but not GEFS+ flies. The distinct alterations of sodium currents in DS and GEFS+ GABAergic interneurons demonstrate that both loss- and gain-of-function alterations in sodium currents are capable of causing reduced repetitive firing and seizure phenotypes. The mutation-specific effects of 5-HTP on heat-induced seizures suggest the serotonin pathway as a potential therapeutic target for DS.

Thomas, R. E., Andrews, L. A., Burman, J. L., Lin, W. Y. and Pallanck, L. J. (2014). PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix. PLoS Genet 10: e1004279. PubMed ID: 24874806
Summary: Loss-of-function mutations in PINK1 (see Drosophila Pink1), which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson's disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, a screen was performed for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. Genetic perturbations targeting the matrix-localized protease Lon were found to cause dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPRmt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPRmt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. These findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria.

Cornelissen, T., Haddad, D., Wauters, F., Van Humbeeck, C., Mandemakers, W., Koentjoro, B., Sue, C., Gevaert, K., De Strooper, B., Verstreken, P. and Vandenberghe, W. (2014). The deubiquitinase USP15 antagonizes Parkin-mediated mitochondrial ubiquitination and mitophagy. Hum Mol Genet [Epub ahead of print]. PubMed ID: 24852371
Summary: Loss-of-function mutations in PARK2, the gene encoding the E3 ubiquitin ligase Parkin, are the most frequent cause of recessive Parkinson's disease (PD). Parkin translocates from the cytosol to depolarized mitochondria, ubiquitinates outer mitochondrial membrane proteins and induces selective autophagy of the damaged mitochondria (mitophagy). This study shows that Ubiquitin-specific protease 15 (USP15), a deubiquitinating enzyme (DUB) widely expressed in brain and other organs, opposes Parkin-mediated mitophagy, while a panel of other DUBs and a catalytically inactive version of USP15 do not. Moreover, knockdown of USP15 rescues the mitophagy defect of PD patient fibroblasts with PARK2 mutations and decreased Parkin levels. USP15 does not affect the ubiquitination status of Parkin or Parkin translocation to mitochondria, but counteracts Parkin-mediated mitochondrial ubiquitination. Knockdown of the DUB CG8334, the closest homolog of USP15 in Drosophila, largely rescues the mitochondrial and behavioural defects of parkin RNAi flies. These data identify USP15 as an antagonist of Parkin and suggest that USP15 inhibition could be a therapeutic strategy for PD cases caused by reduced Parkin levels.

Saturday, June 7th

Xu, S., Tyagi, S. and Schedl, P. (2014). Spermatid Cyst Polarization in Drosophila Depends upon apkc and the CPEB Family Translational Regulator orb2. PLoS Genet 10: e1004380. PubMed ID: 24830287
Summary: Mature Drosophila sperm are highly polarized cells - on one side is a nearly 2 mm long flagellar tail that comprises most of the cell, while on the other is the sperm head, which carries the gamete's genetic information. The polarization of the sperm cells commences after meiosis is complete and the 64-cell spermatid cyst begins the process of differentiation. The spermatid nuclei cluster to one side of the cyst, while the flagellar axonemes grows from the other. The elongating spermatid bundles are also polarized with respect to the main axis of the testis; the sperm heads are always oriented basally, while the growing tails extend apically. This orientation within the testes is important for transferring the mature sperm into the seminal vesicles. Orienting cyst polarization with respect to the main axis of the testis is shown in this study to depend upon atypical Protein Kinase C (aPKC), a factor implicated in polarity decisions in many different biological contexts. When apkc activity is compromised in the male germline, the direction of cyst polarization within this organ is randomized. Significantly, the mechanisms used to spatially restrict apkc activity to the apical side of the spermatid cyst are different from the canonical cross-regulatory interactions between this kinase and other cell polarity proteins that normally orchestrate polarization. The asymmetric accumulation of aPKC protein in the cyst is shown to depend on an mRNA localization pathway that is regulated by the Drosophila CPEB protein Orb2. orb2 is required to properly localize and activate the translation of apkc mRNAs in polarizing spermatid cysts. It was also shown that orb2 functions not only in orienting cyst polarization with respect to the apical-basal axis of the testis, but also in the process of polarization itself. One of the orb2 targets in this process is its own mRNA. Moreover, the proper execution of this orb2 autoregulatory pathway depends upon apkc.

Cai, D., Chen, S. C., Prasad, M., He, L., Wang, X., Choesmel-Cadamuro, V., Sawyer, J. K., Danuser, G. and Montell, D. J. (2014). Mechanical Feedback through E-Cadherin Promotes Direction Sensing during Collective Cell Migration. Cell 157: 1146-1159. PubMed ID: 24855950
Summary: E-cadherin is a major homophilic cell-cell adhesion molecule that inhibits motility of individual cells on matrix. However, its contribution to migration of cells through cell-rich tissues is less clear. An in vivo sensor of mechanical tension across E-cadherin molecules has been developed, which was combined with cell-type-specific RNAi, photoactivatable Rac, and morphodynamic profiling, to interrogate how E-cadherin contributes to collective migration of cells between other cells. Using the Drosophila ovary as a model, it was found that adhesion between border cells and their substrate, the nurse cells, functions in a positive feedback loop with Rac and actin assembly to stabilize forward-directed protrusion and directionally persistent movement. Adhesion between individual border cells communicates direction from the lead cell to the followers. Adhesion between motile cells and polar cells holds the cluster together and polarizes each individual cell. Thus, E-cadherin is an integral component of the guidance mechanisms that orchestrate collective chemotaxis in vivo.

Jia, D., Tamori, Y., Pyrowolakis, G. and Deng, W. M. (2014). Regulation of broad by the Notch pathway affects timing of follicle cell development. Dev Biol [Epub ahead of print]. PubMed ID: 24815210
Summary: During Drosophila oogenesis, activation of Notch signaling in the follicular epithelium (FE) around stage 6 of oogenesis is essential for entry into the endocycle and a series of other changes such as cell differentiation and migration of subsets of the follicle cells. Notch induces the expression of zinc finger protein Hindsight and suppresses homeodomain protein Cut to regulate the mitotic/endocycle (ME) switch. This study reports that broad (br), encoding a small group of zinc-finger transcription factors resulting from alternative splicing, is a transcriptional target of Notch nuclear effector Suppressor of Hairless (Su(H)). The early pattern of Br in the FE, uniformly expressed except in the polar cells, is established by Notch signaling around stage 6, through the binding of Su(H) to the br early enhancer (brE) region. Mutation of the Su(H) binding site leads to a significant reduction ofbrE reporter expression in follicle cells undergoing the endocycle. Chromatin immunoprecipitation results further confirm Su(H) binding to the br early enhancer. Consistent with its expression in follicle cells during midoogenesis, loss of br function results in a delayed entry into the endocycle. These findings suggest an important role of br in the timing of follicle cell development, and its transcriptional regulation by the Notch pathway.

Furriols, M. and Casanova, J. (2014). Germline and somatic vitelline proteins colocalize in aggregates in the follicular epithelium of Drosophila ovaries. Fly (Austin) 8 [Epub ahead of print]. PubMed ID: 24813257
Summary: Nasrat and Polehole, two Drosophila proteins related functionally and by sequence, are secreted from the oocyte and incorporated into the vitelline membrane, where they play a role in the integrity of the same and in the activation of embryonic Torso RTK. In addition, they also accumulate in a punctate pattern in the follicular epithelium. This study shows that their accumulation at the follicle cells depends on their gene expression in the germline, indicating that these proteins move from the oocyte to the follicle cells in a process that does not require endocytosis. Finally cell markers were used to examine the distribution of these proteins at the follicle cells, and they were shown to accumulate in aggregates with vitelline membrane proteins in close association with the plasmatic membrane. It is proposed that these aggregates represent spatially restricted sinks for vitelline membrane proteins that fail to be incorporated into vitelline bodies and later on into the vitelline membrane.

Herzig, B., Yakulov, T. A., Klinge, K., Gunesdogan, U., Jackle, H. and Herzig, A. (2014). Bällchen is required for self-renewal of germline stem cells in Drosophila melanogaster. Biol Open [Epub ahead of print]. PubMed ID: 24876388
Summary: Self-renewing stem cells are pools of undifferentiated cells, which are maintained in cellular niche environments by distinct tissue-specific signalling pathways. In Drosophila melanogaster, female germline stem cells (GSCs) are maintained in a somatic niche of the gonads by BMP signalling. This study reports a novel function of the Drosophila kinase Bällchen (BALL), showing that its cell autonomous role is to maintain the self-renewing capacity of female GSCs independent of BMP signalling. ball mutant GSCs are eliminated from the niche and subsequently differentiate into mature eggs, indicating that BALL is largely dispensable for differentiation. Similar to female GSCs, BALL is required to maintain self-renewal of male GSCs, suggesting a tissue independent requirement of BALL for self-renewal of germline stem cells.

Friday, June 6th

Kassavetis, G. A. and Kadonaga, J. T. (2014). The Annealing Helicase and Branch Migration Activities of Drosophila HARP. PLoS One 9: e98173. PubMed ID: 24866343
Summary: HARP (SMARCAL1, MARCAL1) is an annealing helicase that functions in the repair and restart of damaged DNA replication forks through its DNA branch migration and replication fork regression activities. HARP is conserved among metazoans. HARP from invertebrates differs by the absence of one of the two HARP-specific domain repeats found in vertebrates. The annealing helicase and branch migration activity of invertebrate HARP has not been documented. This study found that HARP from Drosophila melanogaster retains the annealing helicase activity of human HARP, the ability to disrupt D-loops and to branch migrate Holliday junctions, but fails to regress model DNA replication fork structures. A comparison of human and Drosophila HARP on additional substrates revealed that both HARPs are competent in branch migrating a bidirectional replication bubble composed of either DNA:DNA or RNA:DNA hybrid. Human, but not Drosophila, HARP is also capable of regressing a replication fork structure containing a highly stable poly rG:dC hybrid. Persistent RNA:DNA hybrids in vivo can lead to replication fork arrest and genome instability. The ability of HARP to strand transfer hybrids may signify a hybrid removal function for this enzyme, in vivo.

Basquin, D., Spierer, A., Begeot, F., Koryakov, D. E., Todeschini, A. L., Ronsseray, S., Vieira, C., Spierer, P. and Delattre, M. (2014). The Drosophila Su(var)3-7 Gene Is Required for Oogenesis and Female Fertility, Genetically Interacts with piwi and aubergine, but Impacts Only Weakly Transposon Silencing. PLoS One 9: e96802. PubMed ID: 24820312
Summary: Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. This study analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. Evidence is presented that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead to a proposal that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.

Dorogova, N. V., Fedorova, E. V., Bolobolova, E., Ogienko, A. A. and Baricheva, E. M. (2014). GAGA protein is essential for male germ cell development in Drosophila. Genesis [Epub ahead of print]. PubMed ID: 24817547
Summary: The Drosophila Trithorax-like (Trl) gene encodes a GAGA factor which regulates a number of developmentally important genes. This study identified a new function for Drosophila GAGA factor in male germ cell development. Trl mutants carrying strong hypomorphic alleles display loss of primordial germ cells during their migration in embryogenesis and severe disruption in mitochondria structure during early spermatogenesis. The mutation resulted in small testes formation, a deficit of germ cells, abnormal mitochondrial morphogenesis, spermatocyte death through autophagy and partial or complete male sterility. Pleiotropic mutation effects can be explained by the misexpression of GAGA factor target genes, the products of which are required for germ cell progression into mature sperm.

Sorourian, M., Kunte, M. M., Domingues, S., Gallach, M., Ozdil, F., Rio, J. and Betran, E. (2014). Relocation Facilitates the Acquisition of Short cis-Regulatory Regions that Drive the Expression of Retrogenes during Spermatogenesis in Drosophila. Mol Biol Evol [Epub ahead of print]. PubMed ID: 24855141
Summary: Retrogenes are functional processed copies of genes that originate via the retrotranscription of a mRNA intermediate and often exhibit testis-specific expression. Although this expression pattern appears to be favored by selection, the origin of such expression bias remains unexplained. The regulation of two young testis-specific Drosophila retrogenes, Dntf-2r and Pros28.1A, was studied using genetic transformation and the EGFP reporter gene in D. melanogaster. Two different short (<24 bp) regions upstream of the transcription start sites (TSSs) act as testis-specific regulatory motifs in these genes. The Dntf-2r regulatory region is similar to the known β2 tubulin 14 bp testis motif (β2-UE1). Comparative sequence analyses reveal that this motif was already present before the Dntf-2r insertion and was likely driving the transcription of a non-coding RNA. It was also shown that the β2-UE1 occurs in the regulatory regions of other testis-specific retrogenes, and is functional in either orientation. In contrast, the Pros28.1A testes regulatory region in D. melanogaster appears to be novel. Only Pros28.1B, an older paralog of the Pros28.1 gene family, seems to carry a similar regulatory sequence. It is unclear how the Pros28.1A regulatory region was acquired in D. melanogaster, but it might have evolved de novo from within a region that may have been pre-primed for testes expression. We conclude that relocation is critical for the evolutionary origin of male germline-specific cis-regulatory regions of retrogenes because expression depends on either the site of the retrogene insertion or on sequence changes close to the TSS thereafter. As a consequence we infer that positive selection will play a role in the evolution of these regulatory regions and can often act from the moment of the retrocopy insertion.

Thursday, June 5th

Novak, Z. A., Conduit, P. T., Wainman, A. and Raff, J. W. (2014). Asterless Licenses Daughter Centrioles to Duplicate for the First Time in Drosophila Embryos. Curr Biol [Epub ahead of print]. PubMed ID: 24835456
Summary: Centrioles form centrosomes and cilia, and defects in any of these three organelles are associated with human disease. Centrioles duplicate once per cell cycle, when a mother centriole assembles an adjacent daughter during S phase. Daughter centrioles cannot support the assembly of another daughter until they mature into mothers during the next cell cycle. The molecular nature of this daughter-to-mother transition remains mysterious. Pioneering studies in C. elegans identified a set of core proteins essential for centriole duplication, and a similar set have now been identified in other species. The protein kinase ZYG-1/Sak/Plk4 recruits the inner centriole cartwheel components SAS-6 and SAS-5/Ana2/STIL, which then recruit SAS-4/CPAP, which in turn helps assemble the outer centriole microtubules. In flies and humans, the Asterless/Cep152 protein interacts with Sak/Plk4 and Sas-4/CPAP and is required for centriole duplication, although its precise role in the assembly pathway is unclear. This study shows that Asl is not incorporated into daughter centrioles as they assemble during S phase but is only incorporated once mother and daughter separate at the end of mitosis. The initial incorporation of Asterless (Asl) is irreversible, requires DSas-4, and, crucially, is essential for daughter centrioles to mature into mothers that can support centriole duplication. Therefore a 'dual-licensing' model of centriole duplication is proposed, in which Asl incorporation provides a permanent primary license to allow new centrioles to duplicate for the first time, while centriole disengagement provides a reduplication license to allow mother centrioles to duplicate again.

Rubin, T., Karess, R. E. and Rahmani, Z. (2014). Cenp-meta is required for sustained spindle checkpoint. Biol Open. PubMed ID: 24876387
Summary: Cenp-E is a kinesin-like motor protein required for efficient end-on attachment of kinetochores to the spindle microtubules. Cenp-E immunodepletion in Xenopus mitotic extracts results in the loss of mitotic arrest and massive chromosome missegregation, whereas its depletion in mammalian cells leads to chromosome segregation defects despite the presence of a functional spindle assembly checkpoint (SAC). Cenp-meta has previously been reported to be the Drosophila homolog of vertebrate Cenp-E. In this study, it was shown that cenp-metaΔ mutant neuroblasts arrest in mitosis when treated with colchicine. cenp-metaΔ mutant cells display a mitotic delay. Yet, despite the persistence of the two checkpoint proteins Mad2 and BubR1 on unattached kinetochores, these cells eventually enter anaphase and give rise to highly aneuploid daughter cells. Indeed, it was found that cenp-metaΔ mutant cells display a slow but continuous degradation of cyclin B, which eventually triggers the mitotic exit observed. Thus, these data provide evidence for a role of Cenp-meta in sustaining the SAC response.

Wan, L., Tan, M., Yang, J., Inuzuka, H., Dai, X., Wu, T., Liu, J., Shaik, S., Chen, G., Deng, J., Malumbres, M., Letai, A., Kirschner, M. W., Sun, Y. and Wei, W. (2014). APC(Cdc20) Suppresses Apoptosis through Targeting Bim for Ubiquitination and Destruction. Dev Cell 29: 377-391. PubMed ID: 24871945
Summary: Anaphase-promoting complex Cdc20 (APCCdc20; Fizzy in Drosophila) plays pivotal roles in governing mitotic progression. By suppressing APCCdc20, antimitotic agents activate the spindle-assembly checkpoint and induce apoptosis after prolonged treatment, whereas depleting endogenous Cdc20 suppresses tumorigenesis in part by triggering mitotic arrest and subsequent apoptosis. However, the molecular mechanism(s) underlying apoptosis induced by Cdc20 abrogation remains poorly understood. This study reports the BH3-only proapoptotic protein BCL2-like 11 (Bim) as an APCCdc20 target, such that depletion of Cdc20 sensitizes cells to apoptotic stimuli. Strikingly, Cdc20 and multiple APC-core components were identified in a small interfering RNA screen that, upon knockdown, sensitizes otherwise resistant cancer cells to chemoradiation in a Bim-dependent manner. Consistently, human adult T cell leukemia cells that acquire elevated APCCdc20 activity via expressing the Tax viral oncoprotein exhibit reduced Bim levels and resistance to anticancer agents. These results reveal an important role for APCCdc20 in governing apoptosis, strengthening the rationale for developing specific Cdc20 inhibitors as effective anticancer agents.

Hehnly, H. and Doxsey, S. (2014). Rab11 endosomes contribute to mitotic spindle organization and orientation. PubMed ID: 24561039
Summary: During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 endosomes in mitosis. This study, carried out in cultured mammalian cells, shows that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. Mitotic recycling endosomes were shown to bind gamma-TuRC components and associate with tubulin in vitro. Rab11 depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively active Rab11 increases astral microtubules, restores gamma-tubulin spindle pole localization, and generates robust spindles. This suggests a role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. It is proposed that Rab11 and its associated endosomes cocontribute to these processes through retrograde transport to poles by dynein.

Wednesday, June 4th

Kuert, P. A., Hartenstein, V., Bello, B. C., Lovick, J. K. and Reichert, H. (2014). Neuroblast lineage identification and lineage-specific Hox gene action during postembryonic development of the subesophageal ganglion in the Drosophila central brain. Dev Biol 390: 102-115. PubMed ID: 24713419
Summary: The central brain of Drosophila consists of the supraesophageal ganglion (SPG) and the subesophageal ganglion (SEG), both of which are generated by neural stem cell-like neuroblasts during embryonic and postembryonic development. Considerable information has been obtained on postembryonic development of the neuroblasts and their lineages in the SPG. In contrast, very little is known about neuroblasts, neural lineages, or any other aspect of the postembryonic development in the SEG. This study characterized the neuroanatomy of the larval SEG in terms of tracts, commissures, and other landmark features as compared to a thoracic ganglion. Then clonal MARCM labeling was used to identify all adult-specific neuroblast lineages in the late larval SEG, and a surprisingly small number of neuroblast lineages, 13 paired and one unpaired, were found. The Hox genes Dfd, Scr, and Antp are expressed in a lineage-specific manner in these lineages during postembryonic development. Hox gene loss-of-function causes lineage-specific defects in axonal targeting and reduction in neural cell numbers. Moreover, it results in the formation of novel ectopic neuroblast lineages. Apoptosis block also results in ectopic lineages suggesting that Hox genes are required for lineage-specific termination of proliferation through programmed cell death. Taken together, these findings show that postembryonic development in the SEG is mediated by a surprisingly small set of identified lineages and requires lineage-specific Hox gene action to ensure the correct formation of adult-specific neurons in the Drosophila brain.

Shieh, B. H., Kristaponyte, I. and Hong, Y. (2014). Distinct Roles of Arrestin 1 in Photoreceptors During Drosophila Development. J Biol Chem [Epub ahead of print]. PubMed ID: 24838243
Summary: Arrestin regulates many facets of G-protein coupled receptors signaling. In Drosophila, Arrestin 1 (Arr1) is expressed at a lower level than Arrestin 2 (Arr2), and the role of Arr1 in visual physiology is less understood. This study generated transgenic flies expressing enhanced green fluorescent protein tagged Arr1 (Arr1-eGFP) and explored its trafficking in live photoreceptors. Arr1-eGFP is localized in the cytoplasm, and displays light-dependent translocation to the rhabdomere possibly by interacting with photoactivated Rh1 (Rh1*). In the adult, translocation of Arr1-eGFP occurs with slower kinetics when compared to that of Arr2-eGFP. This slower kinetics may be attributable to a reduced level of phosphorylated Rh1*. Indeed, a reduced level of phosphorylated Rh1* recruits a lower level of Arr1-eGFP to rhabdomeres. To investigate whether Arr1 is required for the deactivation of phosphorylated Rh1*, it was shown that in flies with reduced Arr1 prolonged depolarizing afterpotential (PDA) can be triggered with fewer light pulses, indicating that inactivation of phosphorylated Rh1* is compromised when the Arr1 level is reduced. Consistently, Arr1 is no longer required for deactivation of Rh1 in flies expressing phosphorylation deficient Rh1. Previously it was reported that Arr1 displays light-dependent internalization. Unexpectedly, in adult photoreceptors no endocytosis of Arr1-eGFP was observed. In contrast, it was shown that in pupal photoreceptors Arr1-eGFP becomes internalized and sequestered in vesicles within the cytoplasm. Taken together, it is proposed that Arr1 plays distinct roles during development and adulthood. Arr1 orchestrates the recycling of phosphorylated Rh1* in pupae while it regulates the deactivation in adult.

Meyer, S., Schmidt, I. and Klambt, C. (2014). Glia ECM interactions are required to shape the Drosophila nervous system. Mech Dev [Epub ahead of print]. PubMed ID: 24859129
Summary: Organs are characterized by a specific shape that is often remodeled during development. The dynamics of organ shape is in particular evident during the formation of the Drosophila nervous system. During embryonic stages the central nervous system compacts, whereas selective growth occurs during larval stages. The nervous system is covered by a layer of surface glial cells that form the blood brain barrier and a thick extracellular matrix called neural lamella. The size of the neural lamella is dynamically adjusted to the growing nervous system, and this study shows that perineurial glial cells secrete proteases to remodel this matrix. Moreover, an imbalance in proteolytic activity results in an abnormal shape of the nervous system. To identify further components controlling nervous system shape, an RNAi based screen was performed and the gene nolo, which encodes an ADAMTS-like protein, was identified. Loss of function alleles were generated, and a requirement in glial cells was demonstrated. Mutant nolo larvae, however, do not show an abnormal nervous system shape. The only predicted off-target of the nolodsRNA is Oatp30B, which encodes an organic anion transporting protein characterized by an extracellular protease inhibitor domain. Loss of function mutants were generated and double mutant analyses demonstrate a genetic interaction between nolo and Oatp30B which prevented the generation of maternal zygotic mutant larvae.

Hegde, V. R., Vogel, R. and Feany, M. B. (2014). Glia are critical for the neuropathology of complex I deficiency in Drosophila. Hum Mol Genet [Epub ahead of print]. PubMed ID: 24760769
Summary: Mitochondrial electron transport chain (ETC) disorders cause severe neurological disease, typically in the context of fatal encephalomyelopathies. Neuronal cell autonomous energy deficiency due to reduced mitochondrial ATP production is currently the leading hypothesis to explain the neurotoxicity in ETC disorders. To define the mechanisms underlying neuropathology in ETC disorders, this study modeled the most common type of ETC disorder, complex I deficiency, in Drosophila. The model recapitulates important clinical features of the disease including neuronal loss, mitochondrial enlargement, motor dysfunction and early death. Using cell-type specific gene knockdown, it was found that both neurons and glia contribute to the disease phenotype and that glia play a critical non-cell autonomous role in the development of neuronal toxicity. The results open up an unexpected avenue of research, and could lead to the development of new treatment strategies.

Tuesday, June 3rd

DasGupta, S., Ferreira, C. H. and Miesenbock, G. (2014). FoxP influences the speed and accuracy of a perceptual decision in Drosophila. Science 344: 901-904. PubMed ID: 24855268
Summary: Decisions take time if information gradually accumulates to a response threshold, but the neural mechanisms of integration and thresholding are unknown. This study characterized a decision process in Drosophila that bears the behavioral signature of evidence accumulation. As stimulus contrast in trained odor discriminations decreased, reaction times increased and perceptual accuracy declined, in quantitative agreement with a drift-diffusion model. FoxP mutants took longer than wild-type flies to form decisions of similar or reduced accuracy, especially in difficult, low-contrast tasks. RNA interference with FoxP expression in alphabeta core Kenyon cells, or the overexpression of a potassium conductance in these neurons, recapitulated the FoxP mutant phenotype. A mushroom body subdomain whose development or function require the transcription factor FoxP thus supports the progression of a decision toward commitment.

Tuthill, J. C., Nern, A., Rubin, G. M. and Reiser, M. B. (2014). Wide-field feedback neurons dynamically tune early visual processing. Neuron 82: 887-895. PubMed ID: 24853944
Summary: An important strategy for efficient neural coding is to match the range of cellular responses to the distribution of relevant input signals. However, the structure and relevance of sensory signals depend on behavioral state. This study shows that behavior modifies neural activity at the earliest stages of fly vision. A class of wide-field neurons is described that provides feedback to the most peripheral layer of the Drosophila visual system, the lamina. Using in vivo patch-clamp electrophysiology, it was found that lamina wide-field neurons respond to low-frequency luminance fluctuations. Recordings in flying flies revealed that the gain and frequency tuning of wide-field neurons change during flight, and that these effects are mimicked by the neuromodulator octopamine. Genetically silencing wide-field neurons increased behavioral responses to slow-motion stimuli. Together, these findings identify a cell type that is gated by behavior to enhance neural coding by subtracting low-frequency signals from the inputs to motion detection circuits.

Andrews, J. C., Fernandez, M. P., Yu, Q., Leary, G. P., Leung, A. K., Kavanaugh, M. P., Kravitz, E. A. and Certel, S. J. (2014). Octopamine neuromodulation regulates gr32a-linked aggression and courtship pathways in Drosophila males. PLoS Genet 10: e1004356. PubMed ID: 24852170
Summary: Chemosensory pheromonal information regulates aggression and reproduction in many species, but how pheromonal signals are transduced to reliably produce behavior is not well understood. This study demonstrates that the pheromonal signals detected by Gr32a-expressing chemosensory neurons to enhance male aggression are filtered through octopamine (OA, invertebrate equivalent of norepinephrine) neurons. Using behavioral assays, we find males lacking both octopamine and Gr32a gustatory receptors exhibit parallel delays in the onset of aggression and reductions in aggression. Physiological and anatomical experiments identify Gr32a to octopamine neuron synaptic and functional connections in the suboesophageal ganglion. Refining the Gr32a-expressing population indicates that mouth Gr32a neurons promote male aggression and form synaptic contacts with OA neurons. By restricting the monoamine neuron target population, it was shown that three previously identified OA-FruM neurons involved in behavioral choice are among the Gr32a-OA connections. These findings demonstrate that octopaminergic neuromodulatory neurons function as early as a second-order step in this chemosensory-driven male social behavior pathway.

Vasmer, D., Pooryasin, A., Riemensperger, T. and Fiala, A. (2014). Induction of aversive learning through thermogenetic activation of Kenyon cell ensembles in Drosophila. Front Behav Neurosci 8: 174. PubMed ID: 24860455
Summary: Drosophila represents a model organism to analyze neuronal mechanisms underlying learning and memory. Kenyon cells of the Drosophila mushroom body are required for associative odor learning and memory retrieval. But is the mushroom body sufficient to acquire and retrieve an associative memory? To answer this question an experimental approach was conceived to bypass olfactory sensory input and to thermogenetically activate sparse and random ensembles of Kenyon cells directly. It was found that if the artificial activation of Kenyon cell ensembles coincides with a salient, aversive stimulus learning was induced. The animals adjusted their behavior in a subsequent test situation and actively avoided reactivation of these Kenyon cells. These results show that Kenyon cell activity in coincidence with a salient aversive stimulus can suffice to form an associative memory. Memory retrieval is characterized by a closed feedback loop between a behavioral action and the reactivation of sparse ensembles of Kenyon cells.

Bath, D. E., Stowers, J. R., Hormann, D., Poehlmann, A., Dickson, B. J. and Straw, A. D. (2014). FlyMAD: rapid thermogenetic control of neuronal activity in freely walking Drosophila. Nat Methods [Epub ahead of print]. PubMed ID: 24859752
Summary: Rapidly and selectively modulating the activity of defined neurons in unrestrained animals is a powerful approach in investigating the circuit mechanisms that shape behavior. In Drosophila melanogaster, temperature-sensitive silencers and activators are widely used to control the activities of genetically defined neuronal cell types. A limitation of these thermogenetic approaches, however, has been their poor temporal resolution. This study introduced FlyMAD (the fly mind-altering device), which allows thermogenetic silencing or activation within seconds or even fractions of a second. Using computer vision, FlyMAD targets an infrared laser to freely walking flies. As a proof of principle, the rapid silencing and activation of neurons involved in locomotion, vision and courtship were demonstrated. The spatial resolution of the focused beam enabled preferential targeting of neurons in the brain or ventral nerve cord. Moreover, the high temporal resolution of FlyMAD allowed discovery of distinct timing relationships for two neuronal cell types previously linked to courtship song.

Monday, June 2nd

Bhaskar, P. K., Surabhi, S., Tripathi, B. K., Mukherjee, A. and Mutsuddi, M. (2014). dLin52 is crucial for dE2F and dRBF mediated transcriptional regulation of pro-apoptotic gene hid. Biochim Biophys Acta. PubMed ID: 24863159
Summary: Drosophila lin52 (dlin52) is a member of Myb transcription regulator complex and it shows a dynamic pattern of expression in all Drosophila tissues. Myb complex functions to activate or repress transcription in a site-specific manner; however, the detailed mechanism is yet to be clearly understood. Members of the Drosophila melanogaster Myb-MuvB/dREAM complex have been known to regulate expression of a wide range of genes including those involved in regulating apoptosis. E2F and its corepressor RBF also belong to this complex and together they regulate expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. The present study examined whether the depletion of dlin52 in developing photoreceptor neurons results in enhanced apoptosis and disorganisation of the ommatidia. Strikingly, dLin52 was found to be essential for transcriptional repression of the pro-apoptotic gene, hid; decrease in dlin52 levels led to dramatic induction of hid and apoptosis in eye-antennal discs. Reduction of Rpd3 (HDAC1), another member of the dREAM complex, also led to marginal upregulation of Hid. In addition, an optimum level of dLin52 was found to be needed for dE2F1/2 activity on the hid promoter. dlin52 cooperates with dRBF and dE2F1/2 for recruitment of repressor complex on the hid promoter. Preliminary data indicates that Rpd3/HDAC1 also contributes to hid repression. Based on the findings, it is concluded that dLin52 functions as a co-factor and modulates activity of members of dMyb/dREAM complex at hid promoter, thus, regulating apoptosis by repressing this pro-apoptotic gene in the developing Drosophila eye.

Sato-Miyata, Y., Muramatsu, K., Funakoshi, M., Tsuda, M. and Aigaki, T. (2014). Overexpression of dilp2 causes nutrient-dependent semi-lethality in Drosophila. Front Physiol 5: 147. PubMed ID: 24795642
Summary: Insulin/insulin-like growth factor (IGF) plays an important role as a systemic regulator of metabolism in multicellular organisms. Hyperinsulinemia, a high level of blood insulin, is often associated with impaired physiological conditions such as hypoglycemia, insulin resistance, and diabetes. However, due to the complex pathophysiology of hyperinsulinemia, the causative role of excess insulin/IGF signaling has remained elusive. To investigate the biological effects of a high level of insulin in metabolic homeostasis and physiology, flies were generated overexpressing Drosophila insulin-like peptide 2 (Dilp2), which has the highest potential of promoting tissue growth among the Ilp genes in Drosophila. In this model, a UAS-Dilp2 transgene was overexpressed under control of sd-Gal4 that drives expression predominantly in developing imaginal wing discs. Overexpression of Dilp2 caused semi-lethality, which was partially suppressed by mutations in the insulin receptor (InR) or Akt1, suggesting that dilp2-induced semi-lethality is mediated by the PI3K/Akt1 signaling. dilp2-overexpressing flies exhibited intensive autophagy in fat body cells. Interestingly, the dilp2-induced autophagy as well as the semi-lethality was partially rescued by increasing the protein content relative to glucose in the media. These results suggest that excess insulin/IGF signaling impairs the physiology of animals, which can be ameliorated by controlling the nutritional balance between proteins and carbohydrates, at least in flies.

Wang, X., Wang, Z., Chen, Y., Huang, X., Hu, Y., Zhang, R., Ho, M. S. and Xue, L. (2014). FoxO mediates APP-induced AICD-dependent cell death. Cell Death Dis 5: e1233. PubMed ID: 24832605
Summary: The amyloid precursor protein (APP) is a broadly expressed transmembrane protein that has a significant role in the pathogenesis of Alzheimer's disease (AD). APP can be cleaved at multiple sites to generate a series of fragments including the amyloid beta (Abeta) peptides and APP intracellular domain (AICD). Although Abeta peptides have been proposed to be the main cause of AD pathogenesis, the role of AICD has been underappreciated. This study reports that APP induces AICD-dependent cell death in Drosophila neuronal and non-neuronal tissues. A genetic screen identified the transcription factor forkhead box O (FoxO) as a crucial downstream mediator of APP-induced cell death and locomotion defect. In mammalian cells, AICD physically interacts with FoxO in the cytoplasm, translocates with FoxO into the nucleus upon oxidative stress, and promotes FoxO-induced transcription of pro-apoptotic gene Bim. These data demonstrate that APP modulates FoxO-mediated cell death through AICD, which acts as a transcriptional co-activator of FoxO.

Politi, Y., Gal, L., Kalifa, Y., Ravid, L., Elazar, Z. and Arama, E. (2014). Paternal mitochondrial destruction after fertilization is mediated by a common endocytic and autophagic pathway in Drosophila. Dev Cell 29: 305-320. PubMed ID: 24823375
Summary: Almost all animals contain mitochondria of maternal origin only, but the exact mechanisms underlying this phenomenon are still vague. This study investigated the fate of Drosophila paternal mitochondria after fertilization. The sperm mitochondrial derivative (MD) is rapidly eliminated in a stereotypical process dubbed paternal mitochondrial destruction (PMD). PMD is initiated by a network of vesicles resembling multivesicular bodies and displaying common features of the endocytic and autophagic pathways. These vesicles associate with the sperm tail and mediate the disintegration of its plasma membrane. Subsequently, the MD separates from the axoneme and breaks into smaller fragments, which are then sequestered by autophagosomes for degradation in lysosomes. Evidence is provided for the involvement of the ubiquitin pathway and the autophagy receptor p62 in this process. Finally, it was shown that the ubiquitin ligase Parkin is not involved in PMD, implying a divergence from the autophagic pathway of damaged mitochondria.

Lubkov, V. and Bar-Sagi, D. (2014). E-cadherin-mediated cell coupling is required for apoptotic cell extrusion. Curr Biol 24: 868-874. PubMed ID: 24704076
Summary: Apoptotic extrusion is a multicellular process utilized by live cells to remove neighboring apoptotic cells. In epithelial tissues, this process has been shown to be critical for the preservation of tissue integrity and barrier function. This study demonstrates that extrusion is driven by the retraction of the apoptotic cell, which, in turn, triggers a transient and coordinated elongation of the neighboring cells. The coordination of cell elongation requires E-cadherin-mediated cell-cell adhesion. Accordingly, cells that express low levels of E-cadherin (see Drosophila Shotgun) are compromised in elongation and apoptotic extrusion, and furthermore, display loss of barrier function in response to apoptotic stimuli. These findings indicate that the maintenance of adhesive forces during apoptotic cell turnover might play an essential role in controlling tissue homeostasis.

Sunday, June 1st

Rogulja-Ortmann, A., Picao-Osorio, J., Villava, C., Patraquim, P., Lafuente, E., Aspden, J., Thomsen, S., Technau, G. M. and Alonso, C. R. (2014). The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system. Development 141: 2046-2056. PubMed ID: 24803653
Summary: The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. These issues were explored within the developing Drosophila central nervous system (CNS). The pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches it was demonstrated that ELAV binds to discrete elements within Ubx RNAs and that their genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAV-dependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, it is noted that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes it is proposed that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation.

Guilgur, L. G., Prudencio, P., Sobral, D., Liszekova, D., Rosa, A. and Martinho, R. G. (2014). Requirement for highly efficient pre-mRNA splicing during Drosophila early embryonic development. Elife 3: e02181. PubMed ID: 24755291
Summary: Drosophila syncytial nuclear divisions limit transcription unit size of early zygotic genes. As mitosis inhibits not only transcription, but also pre-mRNA splicing, it was reasoned that constraints on splicing were likely to exist in the early embryo; splicing avoidance a possible explanation why most early zygotic genes are intronless. Two mutant alleles were isolated for a subunit of the NTC/Prp19 complex, Fandango, that specifically impaired pre-mRNA splicing of early zygotic but not maternally encoded transcripts. It was hypothesized that the requirements for pre-mRNA splicing efficiency were likely to vary during development. Ectopic maternal expression of an early zygotic pre-mRNA was sufficient to suppress its splicing defects in the mutant background. Furthermore, a small early zygotic transcript with multiple introns was poorly spliced in wild-type embryos. These findings demonstrate for the first time the existence of a developmental prerequisite for highly efficient splicing during Drosophila early embryonic development and suggest a need, in highly proliferative tissues, for coordination between cell cycle and gene architecture to ensure correct gene expression and avoid abnormally processed transcripts.

Williams, S. G. and Hall, K. B. (2014). Linkage and Allostery in snRNP Protein:RNA Complexes. Biochemistry [Epub ahead of print]. PubMed ID: 24849693
Summary: Drosophila SNF is a member of the U1A/U2B''/SNF protein family that is found in U1 and U2 snRNPs, where it binds to Stemloop II and Stemloop IV of U1 or U2 snRNA, respectively. In the U2 snRNP, SNF is also bound to the U2A' protein, which is not found in the U1 snRNP. Although previous reports have implicated U2A' as a necessary auxiliary protein for SNF binding to Stemloop IV, no thermodynamic analysis of the interactions has been done. Using in vitro RNA binding isotherms and isothermal titration calorimetry, the thermodynamics of SNF/RNA/U2A' ternary complex formation have been characterized. There is a very large cooperativity unique to Stemloop IV that explains the localization of U2A' to the U2 snRNP and the exclusion of U2A' from the U1 snRNP. The binding cooperativity, or heterotropic linkage, is interpreted with respect to linked conformational equilibria of both SNF and its RNA ligand. This represents an example of protein-RNA allostery.

Preussner, M., Wilhelmi, I., Schultz, A. S., Finkernagel, F., Michel, M., Moroy, T. and Heyd, F. (2014). Rhythmic U2af26 Alternative Splicing Controls PERIOD1 Stability and the Circadian Clock in Mice. Mol Cell 54: 651-662. PubMed ID: 24837677
Summary: The circadian clock drives daily rhythms in gene expression to control metabolism, behavior, and physiology; while the underlying transcriptional feedback loops are well defined, the impact of alternative splicing on circadian biology remains poorly understood. This paper describes a robust circadian and light-inducible splicing switch that changes the reading frame of the mouse mRNA encoding U2-auxiliary-factor 26 (U2AF26). This results in translation far into the 3' UTR, generating a C terminus with homology to the Drosophila clock regulator Timeless. This new U2AF26 variant destabilizes PERIOD1 protein (see Drosophila Period), and U2AF26-deficient mice show nearly arrhythmic PERIOD1 protein levels and broad defects in circadian mRNA expression in peripheral clocks. At the behavioral level, these mice display increased phase advance adaptation following experimental jet lag. These data suggest light-induced U2af26 alternative splicing to be a buffering mechanism that limits PERIOD1 induction, thus stabilizing the circadian clock against abnormal changes in light:dark conditions.

Algama, M., Oldmeadow, C., Tasker, E., Mengersen, K. and Keith, J. M. (2014). Drosophila 3' UTRs Are More Complex than Protein-Coding Sequences. PLoS One 9: e97336. PubMed ID: 24824035
Summary: The 3' UTRs of eukaryotic genes participate in a variety of post-transcriptional (and some transcriptional) regulatory interactions. Some of these interactions are well characterised, but an undetermined number remain to be discovered. While some regulatory sequences in 3' UTRs may be conserved over long evolutionary time scales, others may have only ephemeral functional significance as regulatory profiles respond to changing selective pressures. This paper proposes a sensitive segmentation methodology for investigating patterns of composition and conservation in 3' UTRs based on comparison of closely related species. Encodings are described of pairwise and three-way alignments integrating information about conservation, GC content and transition/transversion ratios, and the method is applied to three closely related Drosophila species: D. melanogaster, D. simulans and D. yakuba. Incorporating multiple data types greatly increased the number of segment classes identified compared to similar methods based on conservation or GC content alone. It is proposed that the number of segments and number of types of segments identified by the method can be used as proxies for functional complexity. The main finding is that the number of segments and segment classes identified in 3' UTRs is greater than in the same length of protein-coding sequence, suggesting greater functional complexity in 3' UTRs. There is thus a need for sustained and extensive efforts by bioinformaticians to delineate functional elements in this important genomic fraction.

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