What's hot today
Sunday, August 31st, 2014
Vasquez, C. G., Tworoger, M. and Martin, A. C. (2014). Dynamic myosin phosphorylation regulates contractile pulses and tissue integrity during epithelial morphogenesis. J Cell Biol 206: 435-450. PubMed ID: 25092658
Apical constriction is a cell shape change that promotes epithelial bending. Activation of nonmuscle myosin II (Myo-II) by kinases such as Rho-associated kinase (Rok) is important to generate contractile force during apical constriction. Cycles of Myo-II assembly and disassembly, or pulses, are associated with apical constriction during Drosophila melanogaster gastrulation. It is not understood whether Myo-II phosphoregulation organizes contractile pulses or whether pulses are important for tissue morphogenesis. This study shows that Myo-II pulses are associated with pulses of apical Rok. Mutants that mimic Myo-II light chain phosphorylation or depletion of myosin phosphatase inhibit Myo-II contractile pulses, disrupting both actomyosin coalescence into apical foci and cycles of Myo-II assembly/disassembly. Thus, coupling dynamic Myo-II phosphorylation to upstream signals organizes contractile Myo-II pulses in both space and time. Mutants that mimic Myo-II phosphorylation undergo continuous, rather than incremental, apical constriction. These mutants fail to maintain intercellular actomyosin network connections during tissue invagination, suggesting that Myo-II pulses are required for tissue integrity during morphogenesis.
Polyakov, O., He, B., Swan, M., Shaevitz, J. W., Kaschube, M. and Wieschaus, E. (2014). Passive mechanical forces control cell-shape change during Drosophila ventral furrow formation. Biophys J 107: 998-1010. PubMed ID: 25140436
During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. This work develops a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. Novel data is introduced that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. Maintenance of this constant volume is shown to be sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. The specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, this model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that is observed in the actual embryo, with no active force generation required other than apical constriction.
Nowotarski, S. H., McKeon, N., Moser, R. J. and Peifer, M. (2014). The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development. Mol Biol Cell. PubMed ID: 25143400
Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization, and can stimulate filopodia in cultured cells. However, redundancy in mammals and th Diaphanous role in cytokinesis limit analysis of whether and how they regulate protrusions during development. This study used two tissues driving Drosophila dorsal closure, migratory leading-edge (LE) and non-migratory amnioserosal (AS) cells, as models to define how cells shape distinct protrusions during morphogenesis. Non-migratory AS cells were found to produce filopodia that are morphologically and dynamically distinct from those of LE cells. It was hypothesized that differing Enabled and/or Diaphanous activity drive these differences. Combining gain- and loss-of-function with quantitative approaches revealed Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative 'fingerprint', the protrusive profile, which the data suggest is characteristic of each actin regulator. The data suggest LE protrusiveness is primarily Enabled-driven, while Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence.
Kang, M. J., Hansen, T. J., Mickiewicz, M., Kaczynski, T. J., Fye, S. and Gunawardena, S. (2014). Disruption of axonal transport perturbs Bone Morphogenetic Protein (BMP) - signaling and contributes to synaptic abnormalities in rwo neurodegenerative diseases. PLoS One 9: e104617. PubMed ID: 25127478
Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. This study shows that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). Components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility is also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also show synaptic morphological defects. Strikingly TKV motility and p-Mad signaling are disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP; see Drosophila Appl) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins show accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together these results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exists. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases.
Saturday, August 30th
Praveen, K., Wen, Y., Gray, K. M., Noto, J. J., Patlolla, A. R., Van Duyne, G. D. and Matera, A. G. (2014). SMA-Causing Missense Mutations in Survival motor neuron (Smn) Display a Wide Range of Phenotypes When Modeled in Drosophila. PLoS Genet 10: e1004489. PubMed ID: 25144193
Mutations in the human survival motor neuron 1 (SMN; see Drosophila Smn) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, an allelic series was created of twelve Smn missense mutations, originally identified in human SMA patients. Animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN's role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN.
Dusik, V., Senthilan, P. R., Mentzel, B., Hartlieb, H., Wulbeck, C., Yoshii, T., Raabe, T. and Helfrich-Forster, C. (2014). The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock. PLoS Genet 10: e1004565. PubMed ID: 25144774
All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. This study demonstrates by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, it was found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining approximately 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed the Western Blot results and point to p38 as a potential 'clock kinase' phosphorylating Period. Taken together, these findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.
Mizielinska, S., et al. (2014). C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science [Epub ahead of print]. PubMed ID: 25103406
An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question in vitro and in vivo models have been developed to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats in Drosophila caused adult-onset neurodegeneration attributable to poly-(glycine-arginine) proteins. Thus, expanded repeats promoted neurodegeneration through neurotoxic proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence showed both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration.
Saad, Y., Segal, D. and Ayali, A. (2014). Enhanced Neurite Outgrowth and Branching Precede Increased Amyloid-beta-Induced Neuronal Apoptosis in a Novel Alzheimer's Disease Model. J Alzheimers Dis [Epub ahead of print]. PubMed ID: 25125474
Though it is widely accepted that amyloid-β (Aβ; see Drosophila Appl) is a key factor in Alzheimer's disease (AD) pathology, its underling mechanism remains unclear. In order to study the association between Aβ and neural circuitry dysfunction, a primary culture preparation derived from the nervous system of transgenic Drosophila melanogaster larvae was developed expressing human Aβ1-42 (Aβ42). Cultured neurons undergo a consistent developmental process, culminating in an elaborate neuronal network with distinct functional and morphological characteristics. Throughout this development, a time-dependent increase in intracellular expression levels of Aβ42 was detected, followed by extracellular staining at a later time point. When compared to controls, Aβ42 cultures exhibited enhanced levels of apoptosis, resulting in reduced cell viability. Moreover, as primary culture preparations enable high resolution monitoring of neuronal phenotypes, it was possible to detect subtle morphological changes in neurons expressing Aβ42, namely an enhancement in neurite outgrowth and arborization, which preceded the effect of neurodegeneration. These results establish D. melanogaster primary neuronal cultures as a rapid, accessible and cost-effective platform for AD molecular studies and drug screening, and suggest a possible role for Aβ42 in the organization of neuronal processes.
Friday, August 29th
Doamekpor, S. K., Sanchez, A. M., Schwer, B., Shuman, S. and Lima, C. D. (2014). How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes. Genes Dev 28: 1323-1336. PubMed ID: 24939935
Interactions between RNA guanylyltransferase (GTase) and the C-terminal domain (CTD) repeats of RNA polymerase II (see Drosophila Pol2) and elongation factor Spt5 are thought to orchestrate cotranscriptional capping of nascent mRNAs. The crystal structure of a fission yeast GTase*Pol2 CTD complex reveals a unique docking site on the nucleotidyl transferase domain for an 8-amino-acid Pol2 CTD segment, S5PPSYSPTS5P, bracketed by two Ser5-PO4 marks. Analysis of GTase mutations that disrupt the Pol2 CTD interface shows that at least one of the two Ser5-PO4-binding sites is required for cell viability and that each site is important for cell growth at 37 degrees C. Fission yeast GTase binds the Spt5 CTD at a separate docking site in the OB-fold domain that captures the Trp4 residue of the Spt5 nonapeptide repeat T(1)PAW(4)NSGSK. A disruptive mutation in the Spt5 CTD-binding site of GTase is synthetically lethal with mutations in the Pol2 CTD-binding site, signifying that the Spt5 and Pol2 CTDs cooperate to recruit capping enzyme in vivo. CTD phosphorylation has opposite effects on the interaction of GTase with Pol2 (Ser5-PO4 is required for binding) versus Spt5 (Thr1-PO4 inhibits binding). It is proposed that the state of Thr1 phosphorylation comprises a binary 'Spt5 CTD code' that is read by capping enzyme independent of and parallel to its response to the state of the Pol2 CTD.
Lu, C. S., Zhai, B., Mauss, A., Landgraf, M., Gygi, S. and Van Vactor, D. (2014). MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development. Philos Trans R Soc Lond B Biol Sci 369. PubMed ID: 25135978
Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. The localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, this study found that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage it was found that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins.
Afroz, T., Skrisovska, L., Belloc, E., Guillen-Boixet, J., Mendez, R. and Allain, F. H. (2014). A fly trap mechanism provides sequence-specific RNA recognition by CPEB proteins. Genes Dev 28: 1498-1514. PubMed ID: 24990967
Cytoplasmic changes in polyA tail length is a key mechanism of translational control and is implicated in germline development, synaptic plasticity, cellular proliferation, senescence, and cancer progression. The presence of a U-rich cytoplasmic polyadenylation element (CPE) in the 3' untranslated regions (UTRs) of the responding mRNAs gives them the selectivity to be regulated by the CPE-binding (CPEB) family of proteins (see Drosophila Orb), which recognizes RNA via the tandem RNA recognition motifs (RRMs). This study reports the solution structures of the tandem RRMs of two human paralogs (CPEB1 and CPEB4) in their free and RNA-bound states. The structures reveal an unprecedented arrangement of RRMs in the free state that undergo an original closure motion upon RNA binding that ensures high fidelity. Structural and functional characterization of the ZZ domain (zinc-binding domain) of CPEB1 suggests a role in both protein-protein and protein-RNA interactions. Together with functional studies, the structures reveal how RNA binding by CPEB proteins leads to an optimal positioning of the N-terminal and ZZ domains at the 3' UTR, which favors the nucleation of the functional ribonucleoprotein complexes for translation regulation.
Moy, R. H., Cole, B. S., Yasunaga, A., Gold, B., Shankarling, G., Varble, A., Molleston, J. M., tenOever, B. R., Lynch, K. W. and Cherry, S. (2014). Stem-Loop Recognition by DDX17 Facilitates miRNA Processing and Antiviral Defense. Cell 158: 764-777. PubMed ID: 25126784
DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, an evolutionarily conserved and interferon-independent role was identified for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), this study shows that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements.
Thursday, August 28th
Rouault, H., Santolini, M., Schweisguth, F. and Hakim, V. (2014). Imogene: identification of motifs and cis-regulatory modules underlying gene co-regulation. Nucleic Acids Res 42: 6128-6145. PubMed ID: 24682824
Cis-regulatory modules (CRMs) and motifs play a central role in tissue and condition-specific gene expression. This paper presents Imogene, an ensemble of statistical tools that has been developed to facilitate their identification and implemented in a publicly available software. Starting from a small training set of mammalian or fly CRMs that drive similar gene expression profiles, Imogene determines de novo cis-regulatory motifs that underlie this co-expression. It can then predict on a genome-wide scale other CRMs with a regulatory potential similar to the training set. Imogene bypasses the need of large datasets for statistical analyses by making central use of the information provided by the sequenced genomes of multiple species, based on the developed statistical tools and explicit models for transcription factor binding site evolution. Imogene was tested on characterized tissue-specific mouse developmental CRMs. Its ability to identify CRMs with the same specificity based on its de novo created motifs is comparable to that of previously evaluated 'motif-blind' methods. It was further shown, both in flies and in mammals, that Imogene de novo generated motifs are sufficient to discriminate CRMs related to different developmental programs. Notably, purely relying on sequence data, Imogene performs as well in this discrimination task as a previously reported learning algorithm based on Chromatin Immunoprecipitation (ChIP) data for multiple transcription factors at multiple developmental stages.
Ma, L., Quigley, I., Omran, H. and Kintner, C. (2014). Multicilin drives centriole biogenesis via E2f proteins.Genes Dev 28: 14671. PubMed ID: 24934224Summary:
Multiciliate cells employ hundreds of motile cilia to produce fluid flow, which they nucleate and extend by first assembling hundreds of centrioles. In most cells, entry into the cell cycle allows centrioles to undergo a single round of duplication, but in differentiating multiciliate cells, massive centriole assembly occurs in G0 by a process initiated by a small coiled-coil protein, Multicilin. This study shows that Multicilin acts by forming a ternary complex with E2f4 or E2f5 and Dp1 (Drosophila homologs E2f and Dp) that binds and activates most of the genes required for centriole biogenesis, while other cell cycle genes remain off. This complex also promotes the deuterosome pathway of centriole biogenesis by activating the expression of deup1 but not its paralog, cep63. Finally, this study shows that this complex is disabled by mutations in human Multicilin that cause a severe congenital mucociliary clearance disorder due to reduced generation of multiple cilia. By coopting the E2f regulation of cell cycle genes, Multicilin drives massive centriole assembly in epithelial progenitors in a manner required for multiciliate cell differentiation.
Daniel, B., Nagy, G., Hah, N., Horvath, A., Czimmerer, Z., Poliska, S., Gyuris, T., Keirsse, J., Gysemans, C., Van Ginderachter, J. A., Balint, B. L., Evans, R. M., Barta, E. and Nagy, L. (2014). The active enhancer network operated by liganded RXR supports angiogenic activity in macrophages. Genes Dev 28: 1562-1577. PubMed ID: 25030696
RXR signaling (see Drosophila Usp) is predicted to have a major impact in macrophages, but neither the biological consequence nor the genomic basis of its ligand activation is known. Comprehensive genome-wide studies were carried out to map liganded RXR-mediated transcriptional changes, active binding sites, and cistromic interactions in the context of the macrophage genome architecture. The macrophage RXR cistrome has 5200 genomic binding sites, which are not impacted by ligand. Active enhancers are characterized by PU.1 binding, an increase of enhancer RNA, and P300 recruitment. Using these features, 387 liganded RXR-bound enhancers were linked to 226 genes, which predominantly reside in CTCF/cohesin-limited functional domains. These findings were molecularly validated using chromosome conformation capture (3C) and 3C combined with sequencing (3C-seq), and it was also shown that selected long-range enhancers communicate with promoters via stable or RXR-induced loops and that some of the enhancers interact with each other, forming an interchromosomal network. A set of angiogenic genes, including Vegfa, has liganded RXR-controlled enhancers and provides the macrophage with a novel inducible program.
Huang, C. H., Lujambio, A., Zuber, J., Tschaharganeh, D. F., Doran, M. G., Evans, M. J., Kitzing, T., Zhu, N., de Stanchina, E., Sawyers, C. L., Armstrong, S. A., Lewis, J. S., Sherr, C. J. and Lowe, S. W. (2014). CDK9-mediated transcription elongation is required for MYC addiction in hepatocellular carcinoma. Genes Dev 28: 1800-1814. PubMed ID: 25128497
One-year survival rates for newly diagnosed hepatocellular carcinoma (HCC) are <50%, and unresectable HCC carries a dismal prognosis owing to its aggressiveness and the undruggable nature of its main genetic drivers. By screening a custom library of shRNAs directed toward known drug targets in a genetically defined Myc-driven HCC model, cyclin-dependent kinase 9 (Cdk9; see Drosophila Cdk9) was identified as required for disease maintenance. Pharmacological or shRNA-mediated CDK9 inhibition led to robust anti-tumor effects that correlated with MYC (see Drosophila Myc) expression levels and depended on the role that both CDK9 and MYC exert in transcription elongation. These results establish CDK9 inhibition as a therapeutic strategy for MYC-overexpressing liver tumors and highlight the relevance of transcription elongation in the addiction of cancer cells to MYC.
Wednesday, August 27th
>Cragnaz, L., Klima, R., Skoko, N., Budini, M., Feiguin, F. and Baralle, F. E. (2014). Aggregate formation prevents dTDP-43 neurotoxicity in the Drosophila melanogaster eye. Neurobiol Dis [Epub ahead of print]. PubMed ID: 25088712
TDP-43 inclusions are an important histopathological feature in various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis and Fronto-Temporal Lobar Degeneration. However, the relation of these inclusions with the pathogenesis of the disease is still unclear. In fact, the inclusions could be toxic themselves, induce loss of function by sequestering TDP-43 or a combination of both. Previously, a cellular model of aggregation has been developed using the TDP-43 Q/N rich amino acid sequence 331-369 repeated 12 times (12xQ/N), and these cellular inclusions have been shown to be capable of sequestering the endogenous TDP-43 both in non-neuronal and neuronal cells. This model was tested in vivo in the Drosophila melanogaster eye. The eye structure develops normally in the absence of dTDP-43, a fact previously seen in knock out fly strains. Expression of EGFP 12xQ/N does not alter the structure of the eye. In contrast, overexpression of Drosophila TAR DNA-binding protein-43 homolog (TBPH) is neurotoxic and causes necrosis and loss of function of the eye. More important, the neurotoxicity of TBPH can be abolished by its incorporation to the insoluble aggregates induced by EGFP 12xQ/N. This data indicates that aggregation is not toxic per se and instead has a protective role, modulating the functional TBPH available in the tissue. This is an important indication for the possible pathological mechanism in action on ALS patients.
Burman, J. L., Itsara, L. S., Kayser, E. B., Suthammarak, W., Wang, A. M., Kaeberlein, M., Sedensky, M. M., Morgan, P. G. and Pallanck, L. J. (2014). A Drosophila model of mitochondrial disease caused by a complex I mutation that uncouples proton pumping from electron transfer. Dis Model Mech [Epub ahead of print]. PubMed ID: 25085991
Mutations affecting mitochondrial complex I, a multi-subunit assembly that couples electron transfer to proton pumping, are the most frequent cause of heritable mitochondrial diseases. However, the mechanisms by which complex I dysfunction results in disease remain unclear. This study describes a Drosophila model of complex I deficiency caused by a homoplasmic mutation in the mitochondrial-encoded NADH dehydrogenase subunit 2 (ND2) gene. ND2 mutants exhibit phenotypes that resemble symptoms of mitochondrial disease, including shortened lifespan, progressive neurodegeneration, diminished neural mitochondrial membrane potential, and lower levels of neural ATP. Biochemical studies of ND2 mutants reveal that complex I is unable to efficiently couple electron transfer to proton pumping. Thus, this study provides evidence that the ND2 subunit participates directly in the proton pumping mechanism of complex I. Together, these findings support the model that diminished respiratory chain activity, and consequent energy deficiency, are responsible for the pathogenesis of complex I-associated neurodegeneration.
Miura, E., Hasegawa, T., Konno, M., Suzuki, M., Sugeno, N., Fujikake, N., Geisler, S., Tabuchi, M., Oshima, R., Kikuchi, A., Baba, T., Wada, K., Nagai, Y., Takeda, A. and Aoki, M. (2014). VPS35 dysfunction impairs lysosomal degradation of alpha-synuclein and exacerbates neurotoxicity in a Drosophila model of Parkinson's disease. Neurobiol Dis. PubMed ID: 25107340
Mutations in vacuolar protein sorting 35 (VPS35) have been linked to familial Parkinson's disease (PD). VPS35, a component of the retromer, mediates the retrograde transport of cargo from the endosome to the trans-Golgi network. This study shows that retromer depletion increases the lysosomal turnover of the mannose 6-phosphate receptor, thereby affecting the trafficking of cathepsin D (CTSD), a lysosome protease involved in alpha-synuclein (alphaSYN) degradation. VPS35 knockdown perturbed the maturation step of CTSD in parallel with the accumulation of alphaSYN in the lysosomes. Furthermore, it was found that the knockdown of Drosophila VPS35 not only induced the accumulation of the detergent-insoluble alphaSYN species in the brain but also exacerbated both locomotor impairments and mild compound eye disorganization and interommatidial bristle loss in flies expressing human alphaSYN. These findings indicate that the retromer may play a crucial role in alphaSYN degradation by modulating the maturation of CTSD and might thereby contribute to the pathogenesis of the disease.
Hsu, T. C., Wang, C. K., Yang, C. Y., Lee, L. C., Hsieh-Li, H. M., Ro, L. S., Chen, C. M., Lee-Chen, G. J. and Su, M. T. (2014). Deactivation of TBP Contributes to SCA17 Pathogenesis. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25104854
Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant cerebellar ataxia caused by the expansion of polyglutamine (polyQ) within the TATA box-binding protein (TBP). Previous studies have shown that polyQ-expanded TBP forms neurotoxic aggregates and alters downstream genes. However, how expanded polyQ tracts affect the function of TBP and the link between dysfunctional TBP and SCA17 is not clearly understood. In this study a novel Drosophila models was generated for SCA17 that recapitulate pathological features such as aggregate formation, mobility defects, and premature death. In addition to forming neurotoxic aggregates, it was determined that polyQ-expanded TBP reduces its own intrinsic DNA-binding and transcription abilities. Dysfunctional TBP also disrupts normal TBP function. Furthermore, heterozygous dTbp amorph mutant flies exhibited SCA17-like phenotypes and flies expressing polyQ-expanded TBP exhibited enhanced retinal degeneration, suggesting that loss of TBP function may contribute to SCA17 pathogenesis. It was further determined that the downregulation of TBP activity enhances retinal degeneration in SCA3 and Huntington's disease fly models, indicating that the deactivation of TBP is likely to play a common role in polyQ-induced neurodegeneration.
Tuesday, August 26th
Alekseyenko, A. A., Gorchakov, A. A., Zee, B. M., Fuchs, S. M., Kharchenko, P. V. and Kuroda, M. I. (2014). Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs. Genes Dev 28: 1445-1460. PubMed ID: 24990964
Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. This study identifies chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. The results reveal an extensive list of known and novel HP1a-interacting proteins, of which three were selected for validation. A strong novel interactor, dADD1 (Drosophila ADD1) (CG8290), is highly enriched in heterochromatin, harbors an ADD domain similar to human ATRX, displays selective binding to H3K9me2 and H3K9me3, and is a classic genetic suppressor of position-effect variegation. Unexpectedly, a second hit, HIPP1 (HP1 and insulator partner protein-1) (CG3680), is strongly connected to CP190-related complexes localized at putative insulator sequences throughout the genome in addition to its colocalization with HP1a in heterochromatin. A third interactor, the histone methyltransferase MES-4, is also enriched in heterochromatin. In addition to these protein-protein interactions, this study found that HP1a selectively associated with a broad set of RNAs transcribed from repetitive regions. It is proposed that this rich network of previously undiscovered interactions will define how HP1a complexes perform their diverse functions in cells and developing organisms.
Herzog, V. A., Lempradl, A., Trupke, J., Okulski, H., Altmutter, C., Ruge, F., Boidol, B., Kubicek, S., Schmauss, G., Aumayr, K., Ruf, M., Pospisilik, A., Dimond, A., Senergin, H. B., Vargas, M. L., Simon, J. A. and Ringrose, L. (2014). A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element. Nat Genet [Epub ahead of print]. PubMed ID: 25108384
Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation by mechanisms that are poorly understood. This study shows that a switch in forward and reverse noncoding transcription from the Drosophila melanogaster vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both noncoding RNAs inhibit PRC2 histone methyltransferase activity, but, in vivo, only the reverse strand binds PRC2. Overexpression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. It is proposed that the interaction of RNAs with PRC2 is differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of noncoding RNAs occurs at several hundred Polycomb-binding sites in fly and vertebrate genomes. This work identifies a previously unreported and potentially widespread class of PRE/TREs that switch function by switching the direction of noncoding RNA transcription.
Singer, R., Atar, S., Atias, O., Oron, E., Segal, D., Hirsch, J. A., Tuller, T., Orian, A. and Chamovitz, D. A. (2014). Drosophila COP9 signalosome subunit 7 interacts with multiple genomic loci to regulate development. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 25106867
The COP9 signalosome protein complex has a central role in the regulation of development of multicellular organisms. While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression. This study shows, using DamID technology, that COP9 signalosome subunit 7 functionally associates with a large number of genomic loci in the Drosophila genome, and shows that the expression of many genes within these loci is COP9 signalosome-dependent. This association is likely direct as it was shown CSN7 binds DNA in vitro. The genes targeted by CSN7 are preferentially enriched for transcriptionally active regions of the genome, and are involved in the regulation of distinct gene ontology groupings including imaginal disc development and cell-cycle control. In accord, loss of CSN7 function leads to cell-cycle delay and altered wing development. These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control. While the COP9 signalosome protein complex has long been known to regulate protein degradation, this study expands the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health.
Pezic, D., Manakov, S. A., Sachidanandam, R. and Aravin, A. A. (2014). piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells. Genes Dev 28: 1410-1428. PubMed ID: 24939875
Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. This study shows that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments.
Monday, August 25th
>Urban, E., Nagarkar-Jaiswal, S., Lehner, C. F. and Heidmann, S. K. (2014). The Cohesin Subunit Rad21 Is Required for Synaptonemal Complex Maintenance, but Not Sister Chromatid Cohesion, during Drosophila Female Meiosis. PLoS Genet 10: e1004540. PubMed ID: 25101996
Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the alpha-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic alpha-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, this study has analyzed the role of the mitotic Drosophila alpha-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). The lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin.
Afonso, O., Matos, I., Pereira, A. J., Aguiar, P., Lampson, M. A. and Maiato, H. (2014). Feedback control of chromosome separation by a midzone Aurora B gradient. Science 345: 332-336. PubMed ID: 24925910
Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope reassembly (NER). However, how these two processes are coordinated remains unknown. This study, carried out in Drosophila S2 cells, identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases (see Twins) counteracted this gradient and promoted chromosome decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging chromosomes in the main nuclei before completion of NER.
Samel, S. A., Fernandez-Cid, A., Sun, J., Riera, A., Tognetti, S., Herrera, M. C., Li, H. and Speck, C. (2014). A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA. Genes Dev 28: 1653-1666. PubMed ID: 25085418
The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto DNA replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 (see Drosophila Orc1, Cdc6 and Cdt1) assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5 (see Drosophila Mcm5), it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. This study established that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, it was shown that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, it was demonstrated that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. This study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.
Yarosh, W. and Spradling, A. C. (2014). Incomplete replication generates somatic DNA alterations within Drosophila polytene salivary gland cells. Genes Dev 28: 1840-1855. PubMed ID: 25128500
DNA replication remains unfinished in many Drosophila polyploid cells, which harbor disproportionately fewer copies of late-replicating chromosomal regions. By analyzing paired-end high-throughput sequence data from polytene larval salivary gland cells, 112 underreplicated (UR) euchromatic regions were defined, 60-480 kb in size. To determine the effects of underreplication on genome integrity, anomalous read pairs and breakpoint reads were analyzed throughout the euchromatic genome. Each UR euchromatic region contains many different deletions 10-500 kb in size, while very few deletions are present in fully replicated chromosome regions or UR zones from embryo DNA. Thus, during endocycles, stalled forks within UR regions break and undergo local repair instead of remaining stable and generating nested forks. As a result, each salivary gland cell contains hundreds of unique deletions that account for their copy number reductions. Similar UR regions and deletions were observed in ovarian DNA, suggesting that incomplete replication, fork breakage, and repair occur widely in polytene cells. UR regions are enriched in genes encoding immunoglobulin superfamily proteins and contain many neurally expressed and homeotic genes. It is suggested that the extensive somatic DNA instability described in this study underlies position effect variegation, molds the structure of polytene chromosomes, and should be investigated for possible functions.
Sunday, August 24th
Le Thomas, A., Stuwe, E., Li, S., Du, J., Marinov, G., Rozhkov, N., Chen, Y. C., Luo, Y., Sachidanandam, R., Toth, K. F., Patel, D., Aravin, A. A. (2014). Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing. Genes Dev 28: 1667-1680. PubMed ID: 25085419
Small noncoding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. This study found that transgenerationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the histone 3 Lys9 trimethylation (H3K9me3) mark on genomic piRNA cluster sequences. The heterochromatin protein 1 (HP1) homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. It is proposed that transgenerationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels: by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors.
Chen, Y., Boland, A., Kuzuoglu-Ozturk, D., Bawankar, P., Loh, B., Chang, C. T., Weichenrieder, O. and Izaurralde, E. (2014). A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing. Mol Cell 54: 737-750. PubMed ID: 24768540
CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. This study elucidated the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. The conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. It was further shown that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, these data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping.
Malone, C. D., Mestdagh, C., Akhtar, J., Kreim, N., Deinhard, P., Sachidanandam, R., Treisman, J. and Roignant, J. Y. (2014). The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript. Genes Dev 28: 1786-1799. PubMed ID: 25104425
The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex that binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation, and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. This study describes a novel function for the EJC and its splicing subunit, RnpS1, in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where, in the absence of RnpS1, the fourth intron of piwi is retained. This intron contains a weak polypyrimidine tract that is sufficient to confer dependence on RnpS1. Finally, this study demonstrates that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing.
Artieri, C. G. and Fraser, H. B. (2014). Transcript length mediates developmental timing of gene expression across Drosophila. Mol Biol Evol [Epub ahead of print]. PubMed ID: 25069653
The time required to transcribe genes with long primary transcripts may limit their ability to be expressed in cells with short mitotic cycles, a phenomenon termed intron delay. As such short cycles are a hallmark of the earliest stages of insect development, this study tested the impact of intron delay on the Drosophila developmental transcriptome. Long zygotically expressed genes were found to show substantial delay in expression relative to their shorter counterparts, which is not observed for maternally deposited transcripts. Patterns of RNA-seq coverage along transcripts show that this delay is consistent with their inability to completely transcribe long transcripts, but not with transcriptional initiation-based regulatory control. It was further shown that highly expressed zygotic genes maintain compact transcribed regions across the Drosophila phylogeny, allowing conservation of embryonic expression patterns. It is proposed that the physical constraints of intron delay affect patterns of expression and the evolution of gene structure of a substantial portion of the Drosophila transcriptome.
Saturday, August 23rd
Karandikar, U. C., Jin, M., Jusiak, B., Kwak, S., Chen, R. and Mardon, G. (2014). Drosophila eyes absent is required for normal cone and pigment cell development. PLoS One 9: e102143. PubMed ID: 25057928
In Drosophila, development of the compound eye is orchestrated by a network of highly conserved transcriptional regulators known as the retinal determination (RD) network. The retinal determination gene eyes absent (eya) is expressed in most cells within the developing eye field, from undifferentiated retinal progenitors to photoreceptor cells whose differentiation begins at the morphogenetic furrow (MF). Loss of eya expression leads to an early block in retinal development, making it impossible to study the role of eya expression during later steps of retinal differentiation. Two new regulatory regions have been developed that control eya expression during retinal development. These two enhancers are necessary to maintain eya expression anterior to the MF (eya-IAM) and in photoreceptors (eya-PSE), respectively. Deleting these enhancers affects developmental events anterior to the MF as well as retinal differentiation posterior to the MF. In line with previous results, reducing eya expression anterior to the MF was found to affect several early steps during early retinal differentiation, including cell cycle arrest and expression of the proneural gene ato. Consistent with previous observations that suggest a role for eya in cell proliferation during early development, deletion of eya-IAM was found to lead to a marked reduction in the size of the adult retinal field. On the other hand, deletion of eya-PSE leads to defects in cone and pigment cell development. In addition it was found that eya expression is necessary to activate expression of the cone cell marker Cut and to regulate levels of the Hedgehog pathway effector Ci. In summary, this study uncovers novel aspects of eya-mediated regulation of eye development. The genetic tools generated in this study will allow for a detailed study of how the RD network regulates key steps in eye formation.
Guo, Y., Li, Z. and Lin, X. (2014). Hs3st-A and Hs3st-B regulate intestinal homeostasis in Drosophila adult midgut. Cell Signal [Epub ahead of print]. PubMed ID: 25049075
Intrinsic and extrinsic signals as well as the extracellular matrix (ECM) tightly regulate stem cells for tissue homeostasis and regenerative capacity. Little is known about the regulation of tissue homeostasis by the ECM. Heparan sulfate proteoglycans (HSPGs), important components of the ECM, are involved in a variety of biological events. Two heparin sulfate 3-O sulfotransferase (Hs3st) genes, Hs3st-A and Hs3st-B, encode the modification enzymes in heparan sulfate (HS) biosynthesis. This study demonstrates that Hs3st-A and Hs3st-B are required for adult midgut homeostasis. Depletion of Hs3st-A in enterocytes (ECs) results in increased intestinal stem cell (ISC) proliferation and tissue homeostasis loss. Moreover, increased ISC proliferation is also observed in Hs3st-B null mutant alone, or in combination with Hs3st-A RNAi. Hs3st-A depletion-induced ISC proliferation is effectively suppressed by simultaneous inhibition of the EGFR signaling pathway, suggesting that tissue homeostasis loss in Hs3st-A-deficient intestines is due to increased EGFR signaling. Furthermore, this study found that Hs3st-A-depleted ECs are unhealthy and prone to death, while ectopic expression of the antiapoptotic p35 is able to greatly suppress tissue homeostasis loss in these intestines. Together, these data suggest that Drosophila Hs3st-A and Hs3st-B are involved in the regulation of ISC proliferation and midgut homeostasis maintenance.
Yu, S., Nie, Y., Knowles, B., Sakamori, R., Stypulkowski, E., Patel, C., Das, S., Douard, V., Ferraris, R. P., Bonder, E. M., Goldenring, J. R., Ip, Y. T. and Gao, N. (2014). TLR sorting by Rab11 endosomes maintains intestinal epithelial-microbial homeostasis. EMBO J [Epub ahead of print]. PubMed ID: 25063677
Compartmentalization of Toll-like receptors (TLRs) in intestinal epithelial cells (IECs) regulates distinct immune responses to microbes; however, the specific cellular machinery that controls this mechanism has not been fully identified. This study provides genetic evidences that the recycling endosomal compartment in enterocytes maintains a homeostatic TLR9 intracellular distribution, supporting mucosal tolerance to normal microbiota. Genetic ablation of a recycling endosome resident small GTPase, Rab11a (see Drosophila Rab11), a gene adjacent to a Crohn's disease risk locus, in mouse IECs and in Drosophila midgut caused epithelial cell-intrinsic cytokine production, inflammatory bowel phenotype, and early mortality. Unlike wild-type controls, germ-free Rab11a-deficient mouse intestines failed to tolerate the intraluminal stimulation of microbial agonists. Thus, Rab11a endosome controls intestinal host-microbial homeostasis at least partially via sorting TLRs.
He, Q., Wen, D., Jia, Q., Cui, C., Wang, J., Palli, S. R. and Li, S. (2014). Heat shock protein 83 (Hsp83) facilitates Methoprene-tolerant (Met) nuclear import to modulate juvenile hormone signaling. J Biol Chem [Epub ahead of print]. PubMed ID: 25122763
Juvenile hormone (JH) receptors, Met and Gce, transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified the chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with the PAS-B and bHLH domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of the larval fat body cells when the JH titer is low and JH induces Met nuclear import. Hsp83 was also accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuates JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to the reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR.
Friday, August 22nd
Lu, T. Y., Doherty, J. and Freeman, M. R. (2014). DRK/DOS/SOS converge with Crk/Mbc/dCed-12 to activate Rac1 during glial engulfment of axonal debris. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25099352
Nervous system injury or disease leads to activation of glia, which govern postinjury responses in the nervous system. Axonal injury in Drosophila results in transcriptional upregulation of the glial engulfment receptor Draper; there is extension of glial membranes to the injury site (termed activation), and then axonal debris is internalized and degraded. Loss of the small GTPase Rac1 from glia completely suppresses glial responses to injury, but upstream activators remain poorly defined. Loss of the Rac guanine nucleotide exchange factor (GEF) Crk/myoblast city (Mbc)/dCed-12 has no effect on glial activation, but blocks internalization and degradation of debris. This study shows that the signaling molecules Downstream of receptor kinase (DRK) and Daughter of sevenless (DOS) (mammalian homologs, Grb2 and Gab2, respectively) and the GEF Son of sevenless (SOS) (mammalian homolog, mSOS) are required for efficient activation of glia after axotomy and internalization/degradation of axonal debris. At the earliest steps of glial activation, DRK/DOS/SOS function in a partially redundant manner with Crk/Mbc/dCed-12, with blockade of both complexes strongly suppressing all glial responses, similar to loss of Rac1. This work identifies DRK/DOS/SOS as the upstream Rac GEF complex required for glial responses to axonal injury, and demonstrates a critical requirement for multiple GEFs in efficient glial activation after injury and internalization/degradation of axonal debris.
Kwon, J. Y., Dahanukar, A., Weiss, L. A. and Carlson, J. R. (2014). A map of taste neuron projections in the Drosophila CNS. J Biosci 39: 565-574. PubMed ID: 25116611
This paper provides a map of the projections of taste neurons in the CNS of Drosophila. Using a collection of 67 GAL4 drivers representing the entire repertoire of Gr taste receptors, the projections of neurons expressing these drivers was systematically mapped in the thoracico-abdominal ganglion and the suboesophageal ganglion (SOG). Nine categories of projections were mapped in the thoracico-abdominal ganglia and ten categories in the SOG. The projection patterns are modular, and can be interpreted as combinations of discrete pattern elements. The elements can be interpreted in terms of the taste organ from which the projections originate, the structures from which they originate, and the quality of taste information that they represent. The extensive diversity in projection patterns provides an anatomical basis for functional diversity in responses elicited by different taste stimuli.
Lee, E., Jeong, E. H., Jeong, H. J., Yildirim, E., Vanselow, J. T., Ng, F., Liu, Y., Mahesh, G., Kramer, A., Hardin, P. E., Edery, I. and Kim, E. Y. (2014). Phosphorylation of a central clock transcription factor is required for thermal but not photic entrainment. PLoS Genet 10: e1004545. PubMed ID: 25121504
Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK), the master transcription factor driving cyclical gene expression and the rate-limiting component in the Drosophila circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a Drosophila tissue culture system, this study identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A) rescues the arrythmicity of Clkout flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. These findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression.
Forget, A., Bihannic, L., Cigna, S. M., Lefevre, C., Remke, M., Barnat, M., Dodier, S., Shirvani, H., Mercier, A., Mensah, A., Garcia, M., Humbert, S., Taylor, M. D., Lasorella, A. and Ayrault, O. (2014). Shh signaling protects Atoh1 from degradation mediated by the E3 ubiquitin ligase Huwe1 in neural precursors. Dev Cell 29: 649-661. PubMed ID: 24960692
Signaling networks controlled by Sonic hedgehog (SHH; see Drosophila Hedgehog) and the transcription factor Atoh1 (see Drosophila Atonal) regulate the proliferation and differentiation of cerebellar granule neuron progenitors (GNPs). Deregulations in those developmental processes lead to medulloblastoma formation, the most common malignant brain tumor in childhood. Although the protein Atoh1 is a key factor during both cerebellar development and medulloblastoma formation, up-to-date detailed mechanisms underlying its function and regulation have remained poorly understood. This study reports that SHH regulates Atoh1 stability by preventing its phosphodependent degradation by the E3 ubiquitin ligase Huwe1. These results reveal that SHH and Atoh1 contribute to a positive autoregulatory loop promoting neuronal precursor expansion. Consequently, Huwe1 loss in mouse SHH medulloblastoma illustrates the disruption of this developmental mechanism in cancer. Hence, the crosstalk between SHH signaling and Atoh1 during cerebellar development highlights a collaborative network that could be further targeted in medulloblastoma.
Thursday, August 21st
Huang, Y., Wu, Z., Cao, Y., Lang, M., Lu, B. and Zhou, B. (2014). Zinc binding directly regulates Tau toxicity independent of Tau hyperphosphorylation. Cell Rep 8: 831-842. PubMed ID: 25066125
Tau hyperphosphorylation is thought to underlie tauopathy. Working in a Drosophila tauopathy model expressing a human Tau mutant (hTauR406W, or Tau *), this study has shown that zinc contributes to the development of Tau toxicity through two independent actions: by increasing Tau phosphorylation and, more significantly, by directly binding to Tau. Elimination of zinc binding through amino acid substitution of Cys residues has a minimal effect on phosphorylation levels yet essentially eliminates Tau toxicity. The toxicity of the zinc-binding-deficient mutant Tau * (Tau *C2A) and overexpression of native Drosophila Tau, also lacking the corresponding zinc-binding Cys residues, are largely impervious to zinc concentration. Importantly, restoration of zinc-binding ability to Tau * by introduction of a zinc-binding residue (His) into the original Cys positions restores zinc-responsive toxicities in proportion to zinc-binding affinities. These results indicate zinc binding is a substantial contributor to tauopathy and have implications for therapy development.
Bingol, B., Tea, J. S., Phu, L., Reichelt, M., Bakalarski, C. E., Song, Q., Foreman, O., Kirkpatrick, D. S. and Sheng, M. (2014). The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy. Nature 510: 370-375. PubMed ID: 24896179
Cells maintain healthy mitochondria by degrading damaged mitochondria through mitophagy; defective mitophagy is linked to Parkinson's disease. This study reports that USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy driven by the ubiquitin ligase parkin (also known as PARK2; see Drosophila Parkin) and protein kinase PINK1 (see Drosophila Pink1), which are encoded by two genes associated with Parkinson's disease. Parkin ubiquitinates and tags damaged mitochondria for clearance. Overexpression of USP30 removes ubiquitin attached by parkin onto damaged mitochondria and blocks parkin's ability to drive mitophagy, whereas reducing USP30 activity enhances mitochondrial degradation in neurons. Global ubiquitination site profiling identified multiple mitochondrial substrates oppositely regulated by parkin and USP30. Knockdown of USP30 (CG3016) rescues the defective mitophagy caused by pathogenic mutations in parkin and improves mitochondrial integrity in parkin- or PINK1-deficient flies. Knockdown of USP30 in dopaminergic neurons protects flies against paraquat toxicity in vivo, ameliorating defects in dopamine levels, motor function and organismal survival. Thus USP30 inhibition is potentially beneficial for Parkinson's disease by promoting mitochondrial clearance and quality control.
Navarro, J. A., Hessner, S., Yenisetti, S. C., Bayersdorfer, F., Zhang, L., Voigt, A., Schneuwly, S. and Botella, J. A. (2014). Analysis of dopaminergic neuronal dysfunction in genetic and toxin-induced models of Parkinson's disease in Drosophila. J Neurochem [Epub ahead of print]. PubMed ID: 25040725
Drosophila melanogaster has contributed significantly to the understanding of disease mechanisms in Parkinson's disease (PD) as it is one of the very few PD model organisms that allows the study of age-dependent behavioral defects, physiology and histology, and genetic interactions among different PD-related genes. However, there have been contradictory results from a number of recent reports regarding the loss of dopaminergic neurons in different Parkinson's disease fly models. In an attempt to reevaluate and clarify this issue, this study has examined three different genetic (alpha-synuclein, Pink1, parkin) and two toxin-based (rotenone and paraquat) models of the disease for neuronal cell loss. The results showed no dopaminergic neuronal loss in all models tested. Despite this surprising result, this study found additional phenotypes showing the dysfunctional status of the dopaminergic neurons in most of the models analyzed. A common feature found in most models is a quantifiable decrease in the fluorescence of a GFP reporter gene in dopaminergic neurons that correlates well with other phenotypes found for these models and can be reliably used as a hallmark of the neurodegenerative process when modeling diseases affecting the dopaminergic system in Drosophila.
Lopez Del Amo, V., Seco-Cervera, M., Garcia-Gimenez, J. L., Whitworth, A. J., Pallardo, F. V. and Galindo, M. I. (2014). Mitochondrial defects and neuromuscular degeneration caused by altered expression of Drosophila Gdap1: implications for the Charcot-Marie-Tooth neuropathy. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25122658
One of the genes involved in Charcot-Marie-Tooth disease, an inherited peripheral neuropathy, is GDAP1. This work shows that there is a true ortholog of this gene in Drosophila, which has been named Gdap1. By up- and down-regulation of Gdap1 in a tissue-specific manner, this study shows that altering its levels of expression produce changes in mitochondrial size, morphology and distribution; and neuronal and muscular degeneration. Interestingly, muscular degeneration is tissue-autonomous and not dependent on innervation. Metabolic analyses of these experimental genotypes suggest that alterations in oxidative stress are not a primary cause of the neuromuscular degeneration, but a long-term consequence of the underlying mitochondrial dysfunction. The results contribute to a better understanding of the role of mitochondria in Charcot-Marie-Tooth disease, and pave the way to generate clinically relevant disease models to study the relationship between mitochondrial dynamics and peripheral neurodegeneration.
Wednesday, August 20th
Chi, M. W., Griffith, L. C. and Vecsey, C. G. (2014). Larval population density alters adult sleep in wild-type Drosophila melanogaster but not in amnesiac mutant flies. Brain Sci 4: 453-470. PubMed ID: 25116571
Sleep has many important biological functions, but how sleep is regulated remains poorly understood. In humans, social isolation and other stressors early in life can disrupt adult sleep. In fruit flies housed at different population densities during early adulthood, social enrichment was shown to increase subsequent sleep, but it is unknown if population density during early development can also influence adult sleep. To answer this question, Drosophila larvae were maintained at a range of population densities throughout larval development, kept them isolated during early adulthood, and then tested their sleep patterns. The findings reveal that flies that had been isolated as larvae had more fragmented sleep than those that had been raised at higher population densities. This effect was more prominent in females than in males. Larval population density did not affect sleep in female flies that were mutant for amnesiac, which has been shown to be required for normal memory consolidation, adult sleep regulation, and brain development. In contrast, larval population density effects on sleep persisted in female flies lacking the olfactory receptor or83b, suggesting that olfactory signals are not required for the effects of larval population density on adult sleep. These findings show that population density during early development can alter sleep behavior in adulthood, suggesting that genetic and/or structural changes are induced by this developmental manipulation that persist through metamorphosis.
Li-Byarlay, H., Rittschof, C. C., Massey, J. H., Pittendrigh, B. R. and Robinson, G. E. (2014). Socially responsive effects of brain oxidative metabolism on aggression. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25092297
Despite ongoing high energetic demands, brains do not always use glucose and oxygen in a ratio that produces maximal ATP through oxidative phosphorylation. In some cases glucose consumption exceeds oxygen use despite adequate oxygen availability, a phenomenon known as aerobic glycolysis. Although metabolic plasticity seems essential for normal cognition, studying its functional significance has been challenging because few experimental systems link brain metabolic patterns to distinct behavioral states. A recent transcriptomic analysis established a correlation between aggression and decreased whole-brain oxidative phosphorylation activity in the honey bee (Apis mellifera), suggesting that brain metabolic plasticity may modulate this naturally occurring behavior. This study demonstrates that the relationship between brain metabolism and aggression is causal, conserved over evolutionary time, cell type-specific, and modulated by the social environment. Pharmacologically treating honey bees to inhibit complexes I or V in the oxidative phosphorylation pathway resulted in increased aggression. In addition, transgenic RNAi lines and genetic manipulation to knock down gene expression in complex I in fruit fly (Drosophila melanogaster) neurons resulted in increased aggression, but knockdown in glia had no effect. Finally, honey bee colony-level social manipulations that decrease individual aggression attenuated the effects of oxidative phosphorylation inhibition on aggression, demonstrating a specific effect of the social environment on brain function. Because decreased neuronal oxidative phosphorylation is usually associated with brain disease, these findings provide a powerful context for understanding brain metabolic plasticity and naturally occurring behavioral plasticity.
Alves, G., Salle, J., Chaudy, S., Dupas, S. and Maniere, G. (2014). High-NaCl perception in Drosophila melanogaster. J Neurosci 34: 10884-10891. PubMed ID: 25122890
Salt is a fundamental nutrient that is required for many physiological processes, including electrolyte homeostasis and neuronal activity. In mammals and Drosophila, the detection of NaCl induces two different behaviors: low-salt concentrations provide an attractive stimulus, whereas high-salt concentrations are avoided. A gene called serrano (sano) was identified as being expressed in the sensory organs of Drosophila larvae. A transgenic reporter line showed that sano was coexpressed with Gr66a in a subset of gustatory neurons in the terminal organ of third-instar larvae. The disruption of sano gene expression in gustatory neurons led to the specific loss of high-salt concentration avoidance in larvae, whereas the detection of other attractive or aversive substances was unaffected. Moreover, using a cellular marker sensitive to calcium levels, Sano function was shown to be required for neuronal activity in response to high-salt concentrations. In these neurons, the loss of the DEG/ENaC channel PPK19 function also eliminated the cellular response to high-salt concentrations. This study revealed that PPK19 and Sano are required in the neurons of the larval gustatory organs for the detection of high-salt concentrations.
Ramin, M., Domocos, C., Slawaska-Eng, D. and Rao, Y. (2014). Aggression and social experience: genetic analysis of visual circuit activity in the control of aggressiveness in Drosophila. Mol Brain 7: 55. PubMed ID: 25116850
Animal aggressiveness is controlled by genetic and environmental factors. Among environmental factors, social experience plays an important role in modulating aggression in vertebrates and invertebrates. In Drosophila, pheromonal activation of olfactory neurons contributes to social suppression of aggression. While it was reported that impairment in vision decreases the level of aggression in Drosophila, it remains unknown if visual perception also contributes to the modulation of aggression by social experience. This study investigated the role of visual perception in the control of aggression in Drosophila. Several genetic approaches were taken to examine the effects of blocking visual circuit activity on fly aggressive behaviors. In wild type, group housing greatly suppresses aggressiveness. Loss of vision by mutating the ninaB gene does not affect social suppression of fly aggression. Similar suppression of aggressiveness by group housing is observed in fly mutants carrying a mutation in the eya gene leading to complete loss of eyes. Chronic visual loss does not affect the level of aggressiveness of single-housed flies that lack social experience prior to behavioral tests. When visual circuit activity is acutely blocked during behavioral test, however, single-housed flies display higher levels of aggressiveness than that of control flies. It is concluded that visual perception does not play a major role in social suppression of aggression in Drosophila. For single-housed individuals lacking social experience prior to behavioral tests, visual perception decreases the level of aggressiveness.
Seki, Y. and Tanimura, T. (2014). Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila. J Biosci 39: 585-594. PubMed ID: 25116613
A diverse range of organisms shows physiological and behavioural rhythms with various periods. Extensive studies have been performed to elucidate the molecular mechanisms of circadian rhythms with an approximately 24 h period in both Drosophila and mammals, while less attention has been paid to ultradian rhythms with shorter periods. This study used a video-tracking method to monitor the movement of single flies, and clear ultradian rhythms were detected in the locomotor behaviour of wild type and clock mutant flies kept under constant dark conditions. In particular, a Pigment-dispersing factor mutant demonstrated a precise and robust ultradian rhythmicity, which was not temperature compensated. These results suggest that Drosophila has an endogenous ultradian oscillator that is masked by circadian rhythmic behaviours.
Tuesday, August 19th
Demontis, F., Patel, V. K., Swindell, W. R. and Perrimon, N. (2014). Intertissue Control of the Nucleolus via a Myokine-Dependent Longevity Pathway. Cell Rep. PubMed ID: 24882005
Recent evidence indicates that skeletal muscle influences systemic aging, but little is known about the signaling pathways and muscle-released cytokines (myokines) responsible for this intertissue communication. This study shows that muscle-specific overexpression of the transcription factor Mnt decreases age-related climbing defects and extends lifespan in Drosophila. Mnt overexpression in muscle autonomously decreases the expression of nucleolar components and systemically decreases rRNA levels and the size of the nucleolus in adipocytes. This nonautonomous control of the nucleolus, a regulator of ribosome biogenesis and lifespan, relies on Myoglianin, a myokine induced by Mnt and orthologous to human GDF11 and Myostatin. Myoglianin overexpression in muscle extends lifespan and decreases nucleolar size in adipocytes by activating p38 mitogen-activated protein kinase (MAPK), whereas Myoglianin RNAi in muscle has converse effects. Altogether, these findings highlight a key role for myokine signaling in the integration of signaling events in muscle and distant tissues during aging.
Rusan, Z. M., Kingsford, O. A. and Tanouye, M. A. (2014). Modeling Glial Contributions to Seizures and Epileptogenesis: Cation-Chloride Cotransporters in Drosophila melanogaster. PLoS One 9: e101117. PubMed ID: 24971529
Flies carrying a kcc loss-of-function mutation are more seizure-susceptible than wild-type flies. The kcc gene is the highly conserved Drosophila melanogaster ortholog of K+/Cl- cotransporter genes thought to be expressed in all animal cell types. This study examined the spatial and temporal requirements for kcc loss-of-function to modify seizure-susceptibility in flies. Targeted RNA interference (RNAi) of kcc in various sets of neurons was sufficient to induce severe seizure-sensitivity. Interestingly, kcc RNAi in glia was particularly effective in causing seizure-sensitivity. Knockdown of kcc in glia or neurons during development caused a reduction in seizure induction threshold, cell swelling, and brain volume increase in 24-48 hour old adult flies. Third instar larval peripheral nerves were enlarged when kcc RNAi was expressed in neurons or glia. Results suggest that a threshold of K+/Cl- cotransport dysfunction in the nervous system during development is an important determinant of seizure-susceptibility in Drosophila. The findings presented are the first attributing a causative role for glial cation-chloride cotransporters in seizures and epileptogenesis. The importance of elucidating glial cell contributions to seizure disorders and the utility of Drosophila models is discussed.
Park, S., Alfa, R. W., Topper, S. M., Kim, G. E., Kockel, L. and Kim, S. K. (2014). A genetic strategy to measure circulating Drosophila insulin reveals genes regulating insulin production and secretion. PLoS Genet 10: e1004555. PubMed ID: 25101872
Insulin is a major regulator of metabolism in metazoans, including the fruit fly Drosophila melanogaster. Genome-wide association studies (GWAS) suggest a genetic basis for reductions of both insulin sensitivity and insulin secretion, phenotypes commonly observed in humans with type 2 diabetes mellitus (T2DM). To identify molecular functions of genes linked to T2DM risk, a genetic tool was developed to measure insulin-like peptide 2 (Ilp2) levels in Drosophila, a model organism with superb experimental genetics. This system permitted sensitive quantification of circulating Ilp2, including measures of Ilp2 dynamics during fasting and re-feeding, and demonstration of adaptive Ilp2 secretion in response to insulin receptor haploinsufficiency. Tissue specific dissection of this reduced insulin signaling phenotype revealed a critical role for insulin signaling in specific peripheral tissues. Knockdown of the Drosophila orthologues of human T2DM risk genes, including GLIS3 and BCL11A, revealed roles of these Drosophila genes in Ilp2 production or secretion. Discovery of Drosophila mechanisms and regulators controlling in vivo insulin dynamics should accelerate functional dissection of diabetes genetics.
Draper, I., Mahoney, L. J., Mitsuhashi, S., Pacak, C. A., Salomon, R. N. and Kang, P. B. (2014). Silencing of Drpr Leads to Muscle and Brain Degeneration in Adult Drosophila. Am J Pathol [Epub ahead of print]. PubMed ID: 25111228
Mutations in the gene encoding the single transmembrane receptor multiple epidermal growth factor-like domain (MEGF) 10 cause an autosomal recessive congenital muscle disease in humans. Although mammalian MEGF10 is expressed in the central nervous system as well as in skeletal muscle, patients carrying mutations in MEGF10 do not show symptoms of central nervous system dysfunction. Draper (Drpr) is the sole Drosophila homolog of the human genes MEGF10, MEGF11, and MEGF12 (JEDI, PEAR). The functional domains of MEGF10 and Drpr bear striking similarities, and residues affected by MEGF10 mutations in humans are conserved in Drpr. This analysis of drpr mutant flies revealed muscle degeneration with fiber size variability and vacuolization, as well as reduced motor performance, features that have been observed in human MEGF10 myopathy. Vacuolization was also seen in the brain. Tissue-specific RNAi experiments demonstrated that drpr deficiency in muscle, but not in the brain, leads to locomotor defects. The histological and behavioral abnormalities seen in the affected flies set the stage for further studies examining the signaling pathway modulated by MEGF10/Drpr in muscle, as well as assessing the effects of genetic and/or pharmacological manipulations on the observed muscle defects. In addition, the absence of functional redundancy for Drpr in Drosophila may help elucidate whether paralogs of MEGF10 in humans (eg, MEGF11) contribute to maintaining wild-type function in the human brain.
Monday, August 18th
Wang, T., Hauswirth, A. G., Tong, A., Dickman, D. K. and Davis, G. W. (2014). Endostatin is a trans-synaptic signal for homeostatic synaptic plasticity. Neuron 83: 616-629. PubMed ID: 25066085
At synapses in organisms ranging from fly to human, a decrease in postsynaptic neurotransmitter receptor function elicits a homeostatic increase in presynaptic release that restores baseline synaptic efficacy. This process, termed presynaptic homeostasis, requires a retrograde, trans-synaptic signal of unknown identity. Multiplexin was identified in a forward genetic screen for homeostatic plasticity genes. Multiplexin is the Drosophila homolog of Collagen XV/XVIII, a matrix protein that can be proteolytically cleaved to release Endostatin, an antiangiogenesis signaling factor. This study demonstrates that Multiplexin is required for normal calcium channel abundance, presynaptic calcium influx, and neurotransmitter release. Remarkably, Endostatin has a specific activity, independent of baseline synapse development, that is required for the homeostatic modulation of presynaptic calcium influx and neurotransmitter release. These data support a model in which proteolytic release of Endostatin signals trans-synaptically, acting in concert with the presynaptic CaV2.1 calcium channel, to promote presynaptic homeostasis.
Vasin, A., Zueva, L., Torrez, C., Volfson, D., Littleton, J. T. and Bykhovskaia, M. (2014). Synapsin regulates activity-dependent outgrowth of synaptic boutons at the Drosophila neuromuscular junction. J Neurosci 34: 10554-10563. PubMed ID: 25100589Summary:
Patterned depolarization of Drosophila motor neurons can rapidly induce the outgrowth of new synaptic boutons at the larval neuromuscular junction (NMJ), providing a model system to investigate mechanisms underlying acute structural plasticity. Correlative light and electron microscopy analysis revealed that new boutons typically form near the edge of postsynaptic reticulums of presynaptic boutons. Unlike mature boutons, new varicosities have synaptic vesicles which are distributed uniformly throughout the bouton and undeveloped postsynaptic specializations. To characterize the presynaptic mechanisms mediating new synaptic growth induced by patterned activity, the formation of new boutons was investigated in NMJs lacking synapsin [Syn-], a synaptic protein important for vesicle clustering, neurodevelopment, and plasticity. Budding of new boutons at Syn- NMJs was significantly diminished, and new boutons in Syn- preparations were smaller and had reduced synaptic vesicle density. Since synapsin is a target of protein kinase A (PKA), whether activity-dependent synaptic growth is regulated via a cAMP/PKA/synapsin pathway was assayed. Preparations were pretreated with forskolin to raise cAMP levels; this manipulation significantly enhanced activity-dependent synaptic growth in control but not Syn- preparations. To examine the trafficking of synapsin during synaptic growth, transgenic animals were generated expressing fluorescently tagged synapsin. Fluorescence recovery after photobleaching analysis revealed that patterned depolarization promoted synapsin movement between boutons. During new synaptic bouton formation, synapsin redistributed upon stimulation toward the sites of varicosity outgrowth. These findings support a model whereby synapsin accumulates at sites of synaptic growth and facilitates budding of new boutons via a cAMP/PKA-dependent pathway.
Romano, G., Klima, R., Buratti, E., Verstreken, P., Baralle, F. E. and Feiguin, F. (2014). Chronological requirements of TDP-43 function in synaptic organization and locomotive control. Neurobiol Dis. PubMed ID: 25088713
Alterations in TDP-43 are commonly found in patients suffering from amyotrophic lateral sclerosis (ALS) and the genetic suppression of the conserved homologue in Drosophila (TBPH) provokes alterations in the functional organization of motoneuron synaptic terminals, resulting in locomotive defects and reduced life span. To gain more insight into this pathological process, it is of fundamental importance to establish when during the fly life cycle the lack of TBPH affects motoneuron activity and whether this is a reversible phenomenon. To achieve this, the endogenous protein was conditionally expressed in TBPH minus Drosophila neurons, and it was found that TBPH is a short lived protein permanently required for Drosophila motility and synaptic assembly through the direct modulation of vesicular proteins, such as Syntaxin 1A, indicating that synaptic transmission defects are early pathological consequences of TBPH dysfunction in vivo. Importantly, TBPH late induction is able to recover synaptogenesis and locomotion in adult flies revealing an unexpected late-stage functional and structural neuronal plasticity. These observations suggest that late therapeutic approaches based on TDP-43 functionality may also be successful for the human pathology.
Um, K., Niu, S., Duman, J. G., Cheng, J. X., Tu, Y. K., Schwechter, B., Liu, F., Hiles, L., Narayanan, A. S., Ash, R. T., Mulherkar, S., Alpadi, K., Smirnakis, S. M. and Tolias, K. F. (2014). Dynamic control of excitatory synapse development by a Rac1 GEF/GAP regulatory complex. Dev Cell 29: 701-715. PubMed ID: 24960694
The small GTPase Rac1 (see Drosophila Rac1) orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. This study, carried out in cultured mammalian cells, identified a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors (see Drosophila Eph) utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases.
Sunday, August 17th
Dobi, K. C., Halfon, M. S., Baylies, M. K. (2014). Whole-genome analysis of muscle founder cells implicates the chromatin regulator Sin3A in muscle identity. Cell Rep [Epub ahead of print]. PubMed ID: 25088419
Skeletal muscles are formed in numerous shapes and sizes, and this diversity impacts function and disease susceptibility. To understand how muscle diversity is generated, gene expression profiling was performed of two muscle subsets from Drosophila embryos. By comparing the transcriptional profiles of these subsets, a core group of founder cell-enriched genes was identified. Mutants were screened for muscle defects and functions were identified for Sin3A and 10 other transcription and chromatin regulators in the Drosophila embryonic somatic musculature. Sin3A is required for the morphogenesis of a muscle subset, and Sin3A mutants display muscle loss and misattachment. Additionally, misexpression of identity gene transcription factors in Sin3A heterozygous embryos leads to direct transformations of one muscle into another, whereas overexpression of Sin3A results in the reverse transformation. These data implicate Sin3A as a key buffer controlling muscle responsiveness to transcription factors in the formation of muscle identity, thereby generating tissue diversity.
Schottenfeld-Roames, J., Rosa, J. B. and Ghabrial, A. S. (2014). Seamless tube shape is constrained by endocytosis-dependent regulation of active moesin. Curr Biol 24: 1756-1764. PubMed ID: 25065756
Most tubes have seams (intercellular or autocellular junctions that seal membranes together into a tube), but 'seamless' tubes also exist. In Drosophila, stellate-shaped tracheal terminal cells make seamless tubes, with single branches running through each of dozens of cellular extensions. Mutations in braided impair terminal cell branching and cause formation of seamless tube cysts. This study shows that braided encodes Syntaxin7 and that cysts also form in cells deficient for other genes required either for membrane scission (shibire) or for early endosome formation (Rab5, Vps45, and Rabenosyn-5). These data define a requirement for early endocytosis in shaping seamless tube lumens. Importantly, apical proteins Crumbs and phospho-Moesin accumulate to aberrantly high levels in braided terminal cells. Overexpression of either Crumbs or phosphomimetic Moesin induced lumenal cysts and decreased terminal branching. Conversely, the braided seamless tube cyst phenotype was suppressed by mutations in crumbs or Moesin. Indeed, mutations in Moesin dominantly suppressed seamless tube cyst formation and restored terminal branching. It is proposed that early endocytosis maintains normal steady-state levels of Crumbs, which recruits apical phosphorylated (active) Moe, which in turn regulates seamless tube shape through modulation of cortical actin filaments.
Shin, D. H. and Hong, J. W. (2014). Capicua is involved in Dorsal-mediated repression of zerknullt expression in Drosophila embryo. BMB Rep. PubMed ID: 25059278
The maternal transcription factor Dorsal (Dl) functions as both an activator and a repressor in a context-dependent manner to control dorsal-ventral patterning in the Drosophila embryo. Previous studies have suggested that Dl is an intrinsic activator and its repressive activity requires additional corepressors that bind corepressor-binding sites near Dl-binding sites. However, the molecular identities of the corepressors have yet to be identified. This study presents evidence that Capicua (Cic) is involved in Dl-mediated repression in the zerknullt (zen) ventral repression element (VRE). Computational and genetic analyses indicate that a DNA-binding consensus sequence of Cic is highly analogous with previously identified corepressor-binding sequences and that Dl failed to repress zen expression in lateral regions of cic mutant embryos. Furthermore, electrophoretic mobility shift assay (EMSA) shows that Cic directly interacts with several corepressor-binding sites in the zen VRE. These results suggest that Cic may function as a corepressor by binding the VRE.
Umetsu, D., Aigouy, B., Aliee, M., Sui, L., Eaton, S., Julicher, F. and Dahmann, C. (2014). Local increases in mechanical tension shape compartment boundaries by biasing cell intercalations. Curr Biol 24: 1798-1805. PubMed ID: 25065753
Mechanical forces play important roles during tissue organization in developing animals. Many tissues are organized into adjacent, nonmixing groups of cells termed compartments. Boundaries between compartments display a straight morphology and are associated with signaling centers that are important for tissue growth and patterning. Local increases in mechanical tension at cell junctions along compartment boundaries have recently been shown to prevent cell mixing and to maintain straight boundaries. The cellular mechanisms by which local increases in mechanical tension prevent cell mixing at compartment boundaries, however, remain poorly understood. This study has used live imaging and quantitative image analysis to determine cellular dynamics at and near the anteroposterior compartment boundaries of the Drosophila pupal abdominal epidermis. Cell mixing within compartments was shown to involve multiple cell intercalations. Frequency and orientation of cell intercalations are unchanged along the compartment boundaries; rather, an asymmetry in the shrinkage of junctions during intercalation is biased, resulting in cell rearrangements that suppress cell mixing. Simulations of tissue growth show that local increases in mechanical tension can account for this bias in junctional shrinkage. It is concluded that local increases in mechanical tension maintain cell populations separate by influencing junctional rearrangements during cell intercalation.
Saturday, August 16th
Amoyel, M., Simons, B. D. and Bach, E. A. (2014). Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo. EMBO J [Epub ahead of print]. PubMed ID: 25092766
Neutral competition, an emerging feature of stem cell homeostasis, posits that individual stem cells can be lost and replaced by their neighbors stochastically, resulting in chance dominance of a clone at the niche. A single stem cell with an oncogenic mutation could bias this process and clonally spread the mutation throughout the stem cell pool. The Drosophila testis provides an ideal system for testing this model. The niche supports two stem cell populations that compete for niche occupancy. This study shows that cyst stem cells (CySCs) conform to the paradigm of neutral competition and that clonal deregulation of either the Hedgehog (Hh) or Hippo (Hpo) pathway allows a single CySC to colonize the niche. The driving force behind such behavior is accelerated proliferation. These results demonstrate that a single stem cell colonizes its niche through oncogenic mutation by co-opting an underlying homeostatic process.
He, J., Xuan, T., Xin, T., An, H., Wang, J., Zhao, G. and Li, M. (2014). Evidence for Chromatin-Remodeling Complex PBAP-Controlled Maintenance of the Drosophila Ovarian Germline Stem Cells. PLoS One 9: e103473. PubMed ID: 25068272
In the Drosophila oogenesis, germline stem cells (GSCs) continuously self-renew and differentiate into daughter cells for consecutive germline lineage commitment. This developmental process has become an in vivo working platform for studying adult stem cell fate regulation. An increasing number of studies have shown that while concerted actions of extrinsic signals from the niche and intrinsic regulatory machineries control GSC self-renewal and germline differentiation, epigenetic regulation is implicated in the process. This study reports that Brahma (Brm), the ATPase subunit of the Drosophila SWI/SNF chromatin-remodeling complexes, is required for maintaining GSC fate. Removal or knockdown of Brm function in either germline or niche cells causes a GSC loss, but does not disrupt normal germline differentiation within the germarium evidenced at the molecular and morphological levels. There are two Drosophila SWI/SNF complexes: the Brm-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex. More genetic studies reveal that mutations in polybromo/bap180, rather than gene encoding Osa, the BAP complex-specific subunit, elicit a defect in GSC maintenance reminiscent of the brm mutant phenotype. Further genetic interaction test suggests a functional association between brm and polybromo in controlling GSC self-renewal. Taken together, studies in this paper provide the first demonstration that Brm in the form of the PBAP complex functions in the GSC fate regulation.
Li, Y., Ma, Q., Cherry, C. M. and Matunis, E. L. (2014). Steroid signaling promotes stem cell maintenance in the Drosophila testis. Dev Biol [Epub ahead of print]. PubMed ID: 25093968
Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). This study shows that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner Ultraspiracle (Usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex (see Enhancer of bithorax), which has been shown to maintains CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance.
Gaspar, I., Yu, Y. V., Cotton, S. L., Kim, D. H., Ephrussi, A. and Welte, M. A. (2014). Klar ensures thermal robustness of oskar localization by restraining RNP motility. J Cell Biol 206: 199-215. PubMed ID: 25049271
Communication usually applies feedback loop-based filters and amplifiers to ensure undistorted delivery of messages. Such an amplifier acts during Drosophila melanogaster midoogenesis, when oskar messenger ribonucleic acid (mRNA) anchoring depends on its own locally translated protein product. This study found that the motor regulator Klar β mediates a gain-control process that prevents saturation-based distortions in this positive feedback loop. Like oskar mRNA, Klar β localizes to the posterior pole of oocytes in a kinesin-1-dependent manner. By live imaging and semiquantitative fluorescent in situ hybridization, it was shown that Klar β restrains oskar ribonucleoprotein motility and decreases the posterior-ward translocation of oskar mRNA, thereby adapting the rate of oskar delivery to the output of the anchoring machinery. This negative regulatory effect of Klar is particularly important for overriding temperature-induced changes in motility. It is concluded that by preventing defects in oskar anchoring, this mechanism contributes to the developmental robustness of a poikilothermic organism living in a variable temperature environment.
Friday, August 15th
Bowman, S. K., Deaton, A. M., Domingues, H., Wang, P. I., Sadreyev, R. I., Kingston, R. E. and Bender, W. (2014). H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex. Elife (Cambridge): e02833. PubMed ID: 25082344
The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a 'stairstep' pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity.
Shi, J., Zheng, M., Ye, Y., Li, M., Chen, X., Hu, X., Sun, J., Zhang, X. and Jiang, C. (2014). Drosophila Brahma complex remodels nucleosome organizations in multiple aspects. Nucleic Acids Res. PubMed ID: 25081211
ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. Brm was knocked down in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions.
Zhimulev, I. F., Zykova, T. Y., Goncharov, F. P., Khoroshko, V. A., Demakova, O. V., Semeshin, V. F., Pokholkova, G. V., Boldyreva, L. V., Demidova, D. S., Babenko, V. N., Demakov, S. A. and Belyaeva, E. S. (2014). Genetic Organization of Interphase Chromosome Bands and Interbands in Drosophila melanogaster. PLoS One 9: e101631. PubMed ID: 25072930
Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years. This paper analyzed 32 cytology-mapped polytene chromosome interbands. Molecular locations of these interbands was estimated, their molecular and genetic organization was described and it was demonstrated that polytene chromosome interbands contain the 5' ends of housekeeping genes. As a rule, interbands display preferential 'head-to-head' orientation of genes. They are enriched for 'broad' class promoters characteristic of housekeeping genes and associate with open chromatin proteins and Origin Recognition Complex (ORC; see Drosophila Orc) components. In two regions, 10A and 100B, coding sequences of genes whose 5'-ends reside in interbands map to constantly loosely compacted, early-replicating, so-called 'grey' bands. Comparison of expression patterns of genes mapping to late-replicating dense bands vs genes whose promoter regions map to interbands shows that the former are generally tissue-specific, whereas the latter are represented by ubiquitously active genes. Analysis of RNA-seq data (modENCODE-FlyBase) indicates that transcripts from interband-mapping genes are present in most tissues and cell lines studied, across most developmental stages and upon various treatment conditions. A special algorithm was developed to computationally process protein localization data generated by the modENCODE project; it was shown that Drosophila genome has about 5700 sites that demonstrate all the features shared by the interbands cytologically mapped to date.
Hori, T., Shang, W. H., Toyoda, A., Misu, S., Monma, N., Ikeo, K., Molina, O., Vargiu, G., Fujiyama, A., Kimura, H., Earnshaw, W. C. and Fukagawa, T. (2014). Histone H4 Lys 20 monomethylation of the CENP-A nucleosome is essential for kinetochore assembly. Dev Cell 29: 740-749. PubMed ID: 24960696
In vertebrate cells, centromeres are specified epigenetically through the deposition of the centromere-specific histone CENP-A (see Drosophila Centromere identifer). Following CENP-A deposition, additional proteins are assembled on centromeric chromatin. However, it remains unknown whether additional epigenetic features of centromeric chromatin are required for kinetochore assembly. This study used ChIP-seq analysis to examine centromere-specific histone modifications at chicken centromeres, which lack highly repetitive sequences. H4K20 monomethylation (H4K20me1; see Drosophila Histone H4) was found to be enriched at centromeres. Immunofluorescence and biochemical analyses revealed that H4K20me1 is present at all centromeres in chicken and human cells. Based on immunoprecipitation data, H4K20me1 occurs primarily on the histone H4 that is assembled as part of the CENP-A nucleosome following deposition of CENP-A into centromeres. Targeting the H4K20me1-specific demethylase PHF8 to centromeres reduces the level of H4K20me1 at centromeres and results in kinetochore assembly defects. It is concluded that H4K20me1 modification of CENP-A nucleosomes contributes to functional kinetochore assembly.
Thursday, August 14th
Speder, P. and Brand, A. H. (2014). Gap Junction Proteins in the Blood-Brain Barrier Control Nutrient-Dependent Reactivation of Drosophila Neural Stem Cells. Dev Cell. PubMed ID: 25065772
Neural stem cells in the adult brain exist primarily in a quiescent state but are reactivated in response to changing physiological conditions. How do stem cells sense and respond to metabolic changes? In the Drosophila CNS, quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus. Feeding triggers insulin production by blood-brain barrier glial cells, activating the insulin/insulin-like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation. This study shows that gap junction proteins, Inx1 and Inx2, in the blood-brain barrier glia mediate the influence of metabolic changes on stem cell behavior, enabling glia to respond to nutritional signals and reactivate quiescent stem cells. It is proposed that gap junctions in the blood-brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion.
Yeom, E., Hong, S. T. and Choi, K. W. (2014). Crumbs interacts with Xpd for nuclear division control in Drosophila. Oncogene [Epub ahead of print]. PubMed ID: 25065591
Crumbs (Crb) family proteins are crucial for cell polarity. Recent studies indicate that they are also involved in growth regulation and cancer. However, it is not well-understood how Crb participates in mitotic processes. This study reports that Drosophila Crb is critically involved in nuclear division by interacting with Xeroderma pigmentosum D (XPD). A novel gene named galla-1 (CG30152) was identified from a genetic screen for crb modifiers. Galla-1 protein shows homology to MIP18, a subunit of the mitotic spindle-associated MMS19-XPD complex. Loss-of-function galla-1 mutants show abnormal chromosome segregation, defective centrosome positions and branched spindles during nuclear division in early embryos. Embryos with loss-of-function or overexpression of crb show similar mitotic defects and genetic interaction with galla-1. Both Galla-1 and Crb proteins show overlapping localization with spindle microtubules during nuclear division. Galla-1 physically interacts with the intracellular domain of Crb. Interestingly, Galla-1 shows little binding to the Drosophila homolog of XPD, but a related protein Galla-2 (CG7949) binds both Crb and Xpd. Loss-of-function galla-2 mutants show similar mitotic defects as galla-1 and strong genetic interaction with crb. Xpd can form a physical complex with Crb. In imaginal disc, Crb overexpression causes tissue overgrowth as well as DNA damages marked by H2Av phosphorylation. These phenotypes are suppressed by reduction of Xpd. Taken together, this study identifies a novel Crb-Galla-Xpd complex and its function for proper chromosome segregation during nuclear division, implicating a potential link between Crb and Xpd-related genome instability.
Nogueira, C., Kashevsky, H., Pinto, B., Clarke, A. and Orr-Weaver, T. L. (2014). Regulation of Centromere Localization of the Drosophila Shugoshin MEI-S332 and Sister-Chromatid Cohesion in Meiosis. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 25081981
The Shugoshin (Sgo) protein family helps to ensure proper chromosome segregation by protecting cohesion at the centromere by preventing cleavage of the cohesin complex. Some Sgo proteins also influence other aspects of kinetochore-microtubule attachments. Although many Sgo members require Aurora B kinase to localize to the centromere, factors controlling delocalization are poorly understood and diverse. Moreover, it is not clear how Sgo function is inactivated and whether this is distinct from delocalization. This study investigated these questions in Drosophila melanogaster, an organism with superb chromosome cytology to monitor Sgo localization and quantitative assays to test its function in sister-chromatid segregation in meiosis. Previous research showed that in mitosis in cell culture, phosphorylation of the Drosophila Sgo, MEI-S332, by Aurora B promotes centromere localization, whereas Polo phosphorylation promotes delocalization. These studies also suggested that MEI-S332 can be inactivated independently of delocalization, a conclusion supported in this study by localization and function studies in meiosis. Phospho-resistant and phospho-mimetic mutants for the Aurora B and Polo phosphorylation sites were examined for effects on MEI-S332 localization and chromosome segregation in meiosis. Strikingly, MEI-S332 with a phospho-mimetic mutation in the AuroraB phosphorylation site prematurely dissociates from the centromeres in meiosis I. Despite the absence of MEI-S332 on meiosis II centromeres in male meiosis, sister chromatids segregate normally, demonstrating that detectable levels of this Sgo are not essential for chromosome congression, kinetochore biorientation, or spindle assembly.
Fong, C. S., Kim, M., Yang, T. T., Liao, J. C. and Tsou, M. F. (2014). SAS-6 Assembly Templated by the Lumen of Cartwheel-less Centrioles Precedes Centriole Duplication. Dev Cell 30: 238-245. PubMed ID: 25017693
Centrioles are 9-fold symmetric structures duplicating once per cell cycle. Duplication involves self-oligomerization of the centriolar protein SAS-6, but how the 9-fold symmetry is invariantly established remains unclear. This study found that SAS-6 assembly can be shaped by preexisting (or mother) centrioles. During S phase, SAS-6 molecules are first recruited to the proximal lumen of the mother centriole, adopting a cartwheel-like organization through interactions with the luminal wall, rather than via their self-oligomerization activity. The removal or release of luminal SAS-6 requires Plk4 and the cartwheel protein STIL. Abolishing either the recruitment or the removal of luminal SAS-6 hinders SAS-6 (or centriole) assembly at the outside wall of mother centrioles. After duplication, the lumen of engaged mother centrioles becomes inaccessible to SAS-6, correlating with a block for reduplication. These results lead to a proposed model that centrioles may duplicate via a template-based process to preserve their geometry and copy number.
Wednesday August 13th
Dewald, D. N., Steinmetz, E. L. and Walldorf, U. (2014). Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito. Insect Mol Biol [Epub ahead of print]. PubMed ID: 25040100
The Drosophila Homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. This study shows that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and several phosphorylation sites of Nito were mapped. Furthermore, it was possible to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase.
Chou, R. H., et al. (2014). EGFR modulates DNA synthesis and repair through Tyr ohosphorylation of Histone H4. Dev Cell 30: 224-237. PubMed ID: 25073158
Posttranslational modifications of histones play fundamental roles in many biological functions. Specifically, histone H4-K20 (see Drosophila Histone H4) methylation is critical for DNA synthesis and repair. However, little is known about how these functions are regulated by the upstream stimuli. This study identified a tyrosine phosphorylation site at Y72 of histone H4, which facilitates recruitment of histone methyltransferases (HMTases), SET8 and SUV4-20H, to enhance its K20 methylation, thereby promoting DNA synthesis and repair. Phosphorylation-defective histone H4 mutant is deficient in K20 methylation, leading to reduced DNA synthesis, delayed cell cycle progression, and decreased DNA repair ability. Disrupting the interaction between epidermal growth factor receptor (EGFR; see Drosophila EGFR) and histone H4 by Y72 peptide significantly reduced tumor growth. Furthermore, EGFR expression clinically correlates with histone H4-Y72 phosphorylation, H4-K20 monomethylation, and the Ki-67 proliferation marker. These findings uncover a mechanism by which EGFR transduces signal to chromatin to regulate DNA synthesis and repair.
Mahoney, J. E., Mori, M., Szymaniak, A. D., Varelas, X. and Cardoso, W. V. (2014). The hippo pathway effector yap controls patterning and differentiation of airway epithelial progenitors. Dev Cell 30: 137-150. PubMed ID: 25043473
How epithelial progenitor cells integrate local signals to balance expansion with differentiation during organogenesis is still little understood. This study provides evidence that the Hippo pathway effector Yap (see Drosophila Yorkie) is a key regulator of this process in the developing lung. When epithelial tubules are forming and branching, a nucleocytoplasmic shift in Yap localization marks the boundary between the airway and the distal lung compartments. At this transition zone, Yap specifies a transcriptional program that controls Sox2 (see Drosophila SoxNeuro) expression and ultimately generates the airway epithelium. Without Yap, epithelial progenitors are unable to properly respond to local TGF-beta-induced cues and control levels and distribution of Sox2 to form airways. Yap levels and subcellular localization also markedly influence Sox2 expression and differentiation of adult airway progenitors. These data reveal a role for the Hippo-Yap pathway in integrating growth-factor-induced cues in the developing and adult lung potentially key for homeostasis and regeneration repair.
Zhao, R., Fallon, T. R., Saladi, S. V., Pardo-Saganta, A., Villoria, J., Mou, H., Vinarsky, V., Gonzalez-Celeiro, M., Nunna, N., Hariri, L. P., Camargo, F., Ellisen, L. W. and Rajagopal, J. (2014). Yap tunes airway epithelial size and architecture by regulating the identity, maintenance, and self-renewal of stem cells. Dev Cell 30: 151-165. PubMed ID: 25043474
Understanding of how stem cells are regulated to maintain appropriate tissue size and architecture is incomplete. This study shows that Yap (Yes-associated protein 1: see Drosophila Yorkie) is required for the actual maintenance of an adult mammalian stem cell. Without Yap, adult airway basal stem cells are lost through their unrestrained differentiation, resulting in the simplification of a pseudostratified epithelium into a columnar one. Conversely, Yap overexpression increases stem cell self-renewal and blocks terminal differentiation, resulting in epithelial hyperplasia and stratification. Yap overexpression in differentiated secretory cells causes them to partially reprogram and adopt a stem cell-like identity. In contrast, Yap knockdown prevents the dedifferentiation of secretory cells into stem cells. It was then shown that Yap functionally interacts with p63 (see Drosophila p53), the cardinal transcription factor associated with myriad epithelial basal stem cells. In aggregate, this study shows that Yap regulates all of the cardinal behaviors of airway epithelial stem cells and determines epithelial architecture.
Tuesday, August 12th
Kisiel, M., McKenzie, K. and Stewart, B. (2014). Localization and Mobility of Synaptic Vesicles in Myosin VI Mutants of Drosophila. PLoS One 9: e102988. PubMed ID: 25062032
At the Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; however, the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. Myosin VI has been shown to be important for proper synaptic physiology and morphology at the NMJ, likely by functioning as a vesicle tether. This study investigated vesicle dynamics in Myosin VI mutants of Drosophila. In Drosophila, Myosin VI is encoded by the gene, jaguar (jar). To visualize active vesicle cycling FM dye loading was used, and loss of function alleles of jar was compared with controls. These studies revealed a differential distribution of vesicles at the jar mutant nerve terminal, with the newly endocytosed vesicles observed throughout the mutant boutons in contrast to the peripheral localization visualized at control NMJs. This finding is consistent with a role for Myosin VI in restraining vesicle mobility at the synapse to ensure proper localization. To further investigate regulation of vesicle dynamics by Myosin VI, FRAP analysis was used to analyze movement of GFP-labeled synaptic vesicles within individual boutons. FRAP revealed that synaptic vesicles are moving more freely in the jar mutant boutons, indicated by changes in initial bleach depth and rapid recovery of fluorescence following photobleaching. This data provides insights into the role for Myosin VI in mediating synaptic vesicle dynamics at the nerve terminal. Mislocalization of actively cycling vesicles and an apparent increase in vesicle mobility were observed when Myosin VI levels are reduced. These observations support the notion that a major function of Myosin VI in the nerve terminal is tethering synaptic vesicles to proper sub-cellular location within the bouton.
Blunk, A. D., Akbergenova, Y., Cho, R. W., Lee, J., Walldorf, U., Xu, K., Zhong, G., Zhuang, X. and Littleton, J. T. (2014). Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction. Mol Cell Neurosci. PubMed ID: 25066865
Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, a chemical mutagenesis screen was carried out in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen a mutation was identified in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, actE84K, harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous actE84K mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. actE84K mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in actE84K mutants. Genetic interactions between actE84K and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization.
Homma, M., Nagashima, S., Fukuda, T., Yanagi, S., Miyakawa, H., Suzuki, E. and Morimoto, T. (2014). Downregulation of Centaurin gamma1A increases synaptic transmission at Drosophila larval neuromuscular junctions. Eur J Neurosci [Epub ahead of print]. PubMed ID: 25074496
Adequate regulation of synaptic transmission is critical for appropriate neural circuit functioning. Although a number of molecules involved in synaptic neurotransmission have been identified, the molecular mechanisms regulating neurotransmission are not fully understood. This study focused on Centaurin γ1A (CenG1A) and examined its role in synaptic transmission regulation using Drosophila larval neuromuscular junctions. CenG1A is a member of the Centaurin family, which contains Pleckstrin homology, ADP ribosylation factor GTPase-activating protein, and ankyrin repeat domains. Due to the existence of these functional domains, CenG1A is proposed to be involved in the process of synaptic release; however, no evidence for this has been found to date. This study investigated the potential role for CenG1A in the process of synaptic release by performing intracellular recordings in larval muscle cells. Neurotransmitter release from presynaptic cells was found to be enhanced in cenG1A mutants. This effect was also observed in larvae with reduced CenG1A function in either presynaptic or postsynaptic cells. In addition, suppressing CenG1A function in postsynaptic muscle cells leads to an increase in the probability of neurotransmitter release, whereas its suppression in presynaptic neurons leads to an increase in neurotransmitter release probability and an increase in the number of synaptic vesicles. These results suggest that CenG1A functions at both presynaptic and postsynaptic sites as a negative regulator of neurotransmitter release. This study provides evidence for a key role of CenG1A in proper synaptic transmission at neuromuscular junctions.
Fogel, H., Frere, S., Segev, O., Bharill, S., Shapira, I., Gazit, N., O'Malley, T., Slomowitz, E., Berdichevsky, Y., Walsh, D. M., Isacoff, E. Y., Hirsch, J. A. and Slutsky, I. (2014). APP Homodimers Transduce an Amyloid-β-Mediated Increase in Release Probability at Excitatory Synapses. Cell Rep 7: 1560-1576. PubMed ID: 24835997
Accumulation of amyloid-β peptides, the proteolytic products of the amyloid precursor protein (APP, see Drosophila Appl)), induces a variety of synaptic dysfunctions ranging from hyperactivity to depression that are thought to cause cognitive decline in Alzheimer's disease. While depression of synaptic transmission has been extensively studied, the mechanisms underlying synaptic hyperactivity remain unknown. This study shows that Aβ40 monomers and dimers augment release probability through local fine-tuning of APP-APP interactions at excitatory hippocampal boutons. Aβ40 binds to the APP, increases the APP homodimer fraction at the plasma membrane, and promotes APP-APP interactions. The APP activation induces structural rearrangements in the APP/Gi/o-protein complex, boosting presynaptic calcium flux and vesicle release. The APP growth-factor-like domain (GFLD) mediates APP-APP conformational changes and presynaptic enhancement. Thus, the APP homodimer constitutes a presynaptic receptor that transduces signal from Aβ40 to glutamate release. Excessive APP activation may initiate a positive feedback loop, contributing to hippocampal hyperactivity in Alzheimer's disease.
Monday, August 11th
Yashiro, H., Loza, A. J., Skeath, J. B. and Longmore, G. D. (2014). Rho1 Regulates Adherens Junction Remodeling by Promoting Recycling Endosome Formation through Activation of Myosin II. Mol Biol Cell. PubMed ID: 25079692
Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained as AJ are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through DE-cadherin endocytosis by limiting the Cdc42/Par6/aPKC complex activity. This study demonstrates that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin containing Rab11-positive recycling endosomes in Drosophila post-mitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 may regulate the formation of recycling endosomes thorough localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin containing vesicular trafficking during AJ remodeling in live epithelia.
Ukken, F. P., Aprill, I., JayaNandanan, N. and Leptin, M. (2014). Slik and the receptor tyrosine kinase Breathless mediate localized activation of Moesin in terminal tracheal cellsE. PLoS One 9: e103323. PubMed ID: 25061859
A key element in the regulation of subcellular branching and tube morphogenesis of the Drosophila tracheal system is the organization of the actin cytoskeleton by the ERM protein Moesin. Activation of Moesin within specific subdomains of cells, critical for its interaction with actin, is a tightly controlled process and involves regulatory inputs from membrane proteins, kinases and phosphatases. The kinases that activate Moesin in tracheal cells are not known. This study shows that the Sterile-20 like kinase Slik, enriched at the luminal membrane, is necessary for the activation of Moesin at the luminal membrane and regulates branching and subcellular tube morphogenesis of terminal cells. The results reveal the FGF-receptor Breathless as an additional necessary cue for the activation of Moesin in terminal cells. Breathless-mediated activation of Moesin is independent of the canonical MAP kinase pathway.
Hsieh, H. H., Chang, W. T., Yu, L. and Rao, Y. (2014). Control of axon-axon attraction by Semaphorin reverse signaling. Proc Natl Acad Sci U S A. PubMed ID: 25049408
Semaphorin family proteins are well-known axon guidance ligands. Recent studies indicate that certain transmembrane Semaphorins can also function as guidance receptors to mediate axon-axon attraction or repulsion. The mechanisms by which Semaphorin reverse signaling modulates axon-surface affinity, however, remain unknown. This study reveals a novel mechanism underlying upregulation of axon-axon attraction by Semaphorin-1a (Sema1a) reverse signaling in the developing Drosophila visual system. Sema1a promotes the phosphorylation and activation of Moesin (Moe), a member of the ezrin/radixin/moesin family of proteins, and downregulates the level of active Rho1 in photoreceptor axons. It is proposed that Sema1a reverse signaling activates Moe, which in turn upregulates Fas2-mediated axon-axon attraction by inhibiting Rho1.
Wang, Y., Yan, J., Lee, H., Lu, Q. and Adler, P. N. (2014). The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of "upstream" Planar Cell Polarity proteins. Dev Biol. PubMed ID: 25072625
The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins, inturned, fuzzy and fritz, accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, this study has found that when over-expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. It was further shown that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. It was also found that over-expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.
Sunday, August 10th
Kasza, K. E., Farrell, D. L. and Zallen, J. A. (2014). Spatiotemporal control of epithelial remodeling by regulated myosin phosphorylation. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25071215
Spatiotemporally regulated actomyosin contractility generates the forces that drive epithelial cell rearrangements and tissue remodeling. Phosphorylation of the myosin II regulatory light chain (RLC) promotes the assembly of myosin monomers into active contractile filaments and is an essential mechanism regulating the level of myosin activity. However, the effects of phosphorylation on myosin localization, dynamics, and function during epithelial remodeling are not well understood. In Drosophila, planar polarized myosin contractility is required for oriented cell rearrangements during elongation of the body axis. This study shows that regulated myosin phosphorylation influences spatial and temporal properties of contractile behavior at molecular, cellular, and tissue length scales. Expression of myosin RLC variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects at the molecular and cellular levels. Unphosphorylatable RLC produces fewer, slower cell rearrangements, whereas phosphomimetic RLC accelerates rearrangement and promotes higher-order cell interactions. Quantitative live imaging and biophysical approaches reveal that both phosphovariants reduce myosin planar polarity and mechanical anisotropy, altering the orientation of cell rearrangements during axis elongation. Moreover, the localized myosin activator Rho-kinase is required for spatially regulated myosin activity, even when the requirement for phosphorylation is bypassed by the expression of phosphomimetic myosin RLC. These results indicate that myosin phosphorylation influences both the level and the spatiotemporal regulation of myosin activity, linking molecular properties of myosin activity to tissue morphogenesis.
Elkhatib, N., Neu, M. B., Zensen, C., Schmoller, K. M., Louvard, D., Bausch, A. R., Betz, T. and Vignjevic, D. M. (2014). Fascin plays a role in stress fiber organization and focal adhesion disassembly. Curr Biol 24: 1492-1499. PubMed ID: 24930964L
Migrating cells nucleate focal adhesions (FAs) at the cell front and disassemble them at the rear to allow cell translocation. FAs are made of a multiprotein complex, the adhesome, which connects integrins to stress fibers made of mixed-polarity actin filaments. Myosin II-driven contraction of stress fibers generates tensile forces that promote adhesion growth. However, tension must be tightly controlled, because if released, FAs disassemble. Conversely, excess tension can cause abrupt cell detachment resulting in the loss of a major part of the adhesion. Thus, both adhesion growth and disassembly depend on tensile forces generated by stress fiber contraction, but how this contractility is regulated remains unclear. This study shows that the actin-bundling protein Fascin crosslinks the actin filaments into parallel bundles at the stress fibers' termini. Fascin prevents myosin II entry at this region and inhibits its activity in vitro. In fascin-depleted cells, polymerization of actin filaments at the stress fiber termini is slower, the actin cytoskeleton is reorganized into thicker stress fibers with a higher number of myosin II molecules, FAs are larger and less dynamic, and consequently, traction forces that cells exert on their substrate are larger. It was also shown that fascin dissociation from stress fibers is required to allow their severing by cofilin, leading to efficient disassembly of FAs.
Reyes, C. C., Jin, M., Breznau, E. B., Espino, R., Delgado-Gonzalo, R., Goryachev, A. B. and Miller, A. L. (2014). Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin. Curr Biol 24: 1263-1270. PubMed ID: 24835458
Anillin is a scaffolding protein that organizes and stabilizes actomyosin contractile rings and was previously thought to function primarily in cytokinesis. Using Xenopus laevis embryos as a model system to examine Anillin's role in the intact vertebrate epithelium, this study found that a population of Anillin surprisingly localizes to epithelial cell-cell junctions throughout the cell cycle, whereas it was previously thought to be nuclear during interphase. Furthermore, Anillin was shown to play a critical role in regulating cell-cell junction integrity. Both tight junctions and adherens junctions are disrupted when Anillin is knocked down, leading to altered cell shape and increased intercellular spaces. Anillin interacts with Rho, F-actin, and myosin II, all of which regulate cell-cell junction structure and function. When Anillin is knocked down, active Rho (Rho-guanosine triphosphate [GTP]), F-actin, and myosin II are misregulated at junctions. Indeed, increased dynamic 'flares' of Rho-GTP are observed at cell-cell junctions, whereas overall junctional F-actin and myosin II accumulation is reduced when Anillin is depleted. It is proposed that Anillin is required for proper Rho-GTP distribution at cell-cell junctions and for maintenance of a robust apical actomyosin belt, which is required for cell-cell junction integrity. These results reveal a novel role for Anillin in regulating epithelial cell-cell junctions.
Booth, A. J., Blanchard, G. B., Adams, R. J. and Roper, K. (2014). A dynamic microtubule cytoskeleton directs medial actomyosin function during tube formation . Dev Cell 29: 562-576. PubMed ID: 24914560
The cytoskeleton is a major determinant of cell-shape changes that drive the formation of complex tissues during development. Important roles for actomyosin during tissue morphogenesis have been identified, but the role of the microtubule cytoskeleton is less clear. This study shows that during tubulogenesis of the salivary glands in the fly embryo, the microtubule cytoskeleton undergoes major rearrangements, including a 90 degrees change in alignment relative to the apicobasal axis, loss of centrosomal attachment, and apical stabilization. Disruption of the microtubule cytoskeleton leads to failure of apical constriction in placodal cells fated to invaginate. This failure is due to loss of an apical medial actomyosin network whose pulsatile behavior in wild-type embryos drives the apical constriction of the cells. The medial actomyosin network interacts with the minus ends of acentrosomal microtubule bundles through the cytolinker protein Shot, and disruption of Shot also impairs apical constriction.
Saturday, August 9th
Baumgardt, M., Karlsson, D., Salmani, B. Y., Bivik, C., MacDonald, R. B., Gunnar, E. and Thor, S. (2014). Global programmed switch in neural daughter cell proliferation mode triggered by a temporal gene cascade. Dev Cell 30: 192-208. PubMed ID: 25073156
During central nervous system (CNS) development, progenitors typically divide asymmetrically, renewing themselves while budding off daughter cells with more limited proliferative potential. Variation in daughter cell proliferation has a profound impact on CNS development and evolution, but the underlying mechanisms remain poorly understood. This study found that Drosophila embryonic neural progenitors (neuroblasts) undergo a programmed daughter proliferation mode switch, from generating daughters that divide once (type I) to generating neurons directly (type 0). This typeI>0 switch is triggered by activation of Dacapo (mammalian p21(CIP1)/p27(KIP1)/p57(Kip2)) expression in neuroblasts. In the thoracic region, Dacapo expression is activated by the temporal cascade (castor) and the Hox gene Antennapedia. In addition, castor, Antennapedia, and the late temporal gene grainyhead act combinatorially to control the precise timing of neuroblast cell-cycle exit by repressing Cyclin E and E2f. This reveals a logical principle underlying progenitor and daughter cell proliferation control in the Drosophila CNS.
Zhu, Q., Song, L., Peng, G., Sun, N., Chen, J., Zhang, T., Sheng, N., Tang, W., Qian, C., Qiao, Y., Tang, K., Han, J. D., Li, J., Jing, N. (2014). The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways. Elife (Cambridge): e02224. PubMed ID: 24929964
The neural fate commitment of pluripotent stem cells requires the repression of extrinsic inhibitory signals and the activation of intrinsic positive transcription factors. However, how these two events are integrated to ensure appropriate neural conversion remains unclear. This study showed that Pou3f1 is essential for the neural differentiation of mouse embryonic stem cells (ESCs), specifically during the transition from epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs). Chimeric analysis showed that Pou3f1 knockdown leads to a markedly decreased incorporation of ESCs in the neuroectoderm. By contrast, Pou3f1-overexpressing ESC derivatives preferentially contribute to the neuroectoderm. Genome-wide ChIP-seq and RNA-seq analyses indicated that Pou3f1 is an upstream activator of neural lineage genes, and also is a repressor of BMP and Wnt signaling. These results established that Pou3f1 promotes the neural fate commitment of pluripotent stem cells through a dual role, activating internal neural induction programs and antagonizing extrinsic neural inhibitory signals.
Petrovic, J., Formosa-Jordan, P., Luna-Escalante, J. C., Abello, G., Ibanes, M., Neves, J. and Giraldez, F. (2014). Ligand-dependent Notch signaling strength orchestrates lateral induction and lateral inhibition in the developing inner ear. Development 141: 2313-2324. PubMed ID: 24821984
During inner ear development, Notch (see Drosophila Notch) exhibits two modes of operation: lateral induction, which is associated with prosensory specification, and lateral inhibition, which is involved in hair cell determination. These mechanisms depend respectively on two different ligands, jagged 1 (Jag1) and delta 1 (Dl1) (see Drosophila Delta), that rely on a common signaling cascade initiated after Notch activation. In the chicken otocyst, expression of Jag1 and the Notch target Hey1 (see Drosophila Hairy) correlates well with lateral induction, whereas both Jag1 and Dl1 are expressed during lateral inhibition, as are Notch targets Hey1 and Hes5. This study shows that Jag1 drives lower levels of Notch activity than Dl1, which results in the differential expression of Hey1 and Hes5. In addition, Jag1 interferes with the ability of Dl1 to elicit high levels of Notch activity. Modeling the sensory epithelium when the two ligands are expressed together shows that ligand regulation, differential signaling strength and ligand competition are crucial to allow the two modes of operation and for establishing the alternate pattern of hair cells and supporting cells. Jag1, while driving lateral induction on its own, facilitates patterning by lateral inhibition in the presence of Dl1. This novel behavior emerges from Jag1 acting as a competitive inhibitor of Dl1 for Notch signaling. Both modeling and experiments show that hair cell patterning is very robust. The model suggests that autoactivation of proneural factor Atoh1 (see Drosophila Atonal), upstream of Dl1, is a fundamental component for robustness. The results stress the importance of the levels of Notch signaling and ligand competition for Notch function.
Gao, Y., Toska, E., Denmon, D., Roberts, S. G. and Medler, K. F. (2014). WT1 regulates the development of the posterior taste field. Development 141: 2271-2278. PubMed ID: 24803588
Despite the importance of taste in determining nutrient intake, understanding of the processes that control the development of the peripheral taste system is lacking. Several early regulators of taste development have been identified, including sonic hedgehog, bone morphogenetic protein 4 and multiple members of the Wnt/β-catenin signaling pathway. However, the regulation of these factors, including their induction, remains poorly understood. This study identified a crucial role for the Wilms' tumor 1 protein (WT1; see Drosophila Klumpfuss) in circumvallate (CV) papillae development. WT1 is a transcription factor that is important in the normal development of multiple tissues, including both the olfactory and visual systems. In mice, WT1 expression is detectable by E12.5, when the CV taste placode begins to form. In mice lacking WT1, the CV fails to develop normally and markers of early taste development are dysregulated compared with wild type. Expression of the WT1 target genes Lef1, Ptch1 and Bmp4 (see Drosophila Pangolin, Patched, and Decapentaplegic) is significantly reduced in developing tongue tissue derived from Wt1 knockout mice, and, in normal tongue, WT1 was shown to be bound to the promoter regions of these genes. Moreover, siRNA knockdown of WT1 in cultured taste cells leads to a reduction in the expression of Lef1 and Ptch1. These data identify WT1 as a crucial transcription factor in the development of the CV through the regulation of multiple signaling pathways that have established roles in the formation and patterning of taste placodes.
Friday, August 8th
Truscott, M., Islam, A. B., Lightfoot, J., Lopez-Bigas, N. and Frolov, M. V. (2014). An intronic microRNA links Rb/E2F and EGFR signaling. PLoS Genet 10: e1004493. PubMed ID: 25058496
The importance of microRNAs in the regulation of various aspects of biology and disease is well recognized. However, what remains largely unappreciated is that a significant number of miRNAs are embedded within and are often co-expressed with protein-coding host genes. Such a configuration raises the possibility of a functional interaction between a miRNA and the gene it resides in. This is exemplified by the Drosophila melanogaster dE2f1 gene that harbors two miRNAs, mir-11 and mir-998, within its last intron. miR-11 was demonstrated to limit the proapoptotic function of dE2F1 by repressing cell death genes that are directly regulated by dE2F1, however the biological role of miR-998 was unknown. This study shows that one of the functions of miR-998 is to suppress dE2F1-dependent cell death specifically in rbf mutants by elevating EGFR signaling. Mechanistically, miR-998 operates by repressing dCbl, a negative regulator of EGFR signaling. Significantly, dCbl is a critical target of miR-998 since dCbl phenocopies the effects of miR-998 on dE2f1-dependent apoptosis in rbf mutants. Importantly, this regulation is conserved, as the miR-998 seed family member miR-29 repressed c-Cbl, and enhanced MAPK activity and wound healing in mammalian cells. Therefore, the two intronic miRNAs embedded in the dE2f1 gene limit the apoptotic function of dE2f1, but operate in different contexts and act through distinct mechanisms. These results also illustrate that examining an intronic miRNA in the context of its host's function can be valuable in elucidating the biological function of the miRNA, and provide new information about the regulation of the host gene itself.
Fagegaltier, D., Konig, A., Gordon, A., Lai, E. C., Gingeras, T. R., Hannon, G. J. and Shcherbata, H. R. (2014). A genome-wide survey of sexually dimorphic expression of Drosophila miRNAs identifies the steroid hormone-induced miRNA let-7 as a regulator of sexual identity. Genetics [Epub ahead of print]. PubMed ID: 25081570
miRNAs bear an increasing number of functions throughout development and in the aging adult. This study addresses their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. A survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. The male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. This study set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone induced let-7 result in aberrant gonadal somatic cell behavior and non cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex determination genes persist in aging adults under hormonal control. Together, findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that mechanisms similar to vertebrate hormonal control of cellular sexual identity exist in Drosophila.
Hayashi, R., Handler, D., Ish-Horowicz, D. and Brennecke, J. (2014). The exon junction complex is required for definition and excision of neighboring introns in Drosophila. Genes Dev [Epub ahead of print]. PubMed ID: 25081352
Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. This study shows that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, it is proposed that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events.
Huang, H., Li, Y., Szulwach, K. E., Zhang, G., Jin, P. and Chen, D. (2014). AGO3 Slicer activity regulates mitochondria-nuage localization of Armitage and piRNA amplification. J Cell Biol 206: 217-230. PubMed ID: 25049272
In Drosophila melanogaster the reciprocal 'Ping-Pong' cycle of PIWI-interacting RNA (piRNA)-directed RNA cleavage catalyzed by the endonuclease (or 'Slicer') activities of the PIWI proteins Aubergine (Aub) and Argonaute3 (AGO3) has been proposed to expand the secondary piRNA population. However, the role of AGO3/Aub Slicer activity in piRNA amplification remains to be explored. This study shows that AGO3 Slicer activity is essential for piRNA amplification and that AGO3 inhibits the homotypic Aub:Aub Ping-Pong process in a Slicer-independent manner. It was also found that expression of an AGO3 Slicer mutant causes ectopic accumulation of Armitage, a key component in the primary piRNA pathway, in the Drosophila melanogaster germline granules known as nuage. AGO3 also coexists and interacts with Armitage in the mitochondrial fraction. Furthermore, AGO3 acts in conjunction with the mitochondria-associated protein Zucchini to control the dynamic subcellular localization of Armitage between mitochondria and nuage in a Slicer-dependent fashion. Collectively, these findings uncover a new mechanism that couples mitochondria with nuage to regulate secondary piRNA amplification.
Thursday, August 7th
Gold, K. S. and Brand, A. H. (2014). Optix defines a neuroepithelial compartment in the optic lobe of the Drosophila brain. Neural Dev 9: 18. PubMed ID: 25074684
During early brain development, the organisation of neural progenitors into a neuroepithelial sheet maintains tissue integrity during growth. Neuroepithelial cohesion and patterning is essential for orderly proliferation and neural fate specification. Neuroepithelia are regionalised by the expression of transcription factors and signalling molecules, resulting in the formation of distinct developmental, and ultimately functional, domains. This study discovered that the Six3/6 family orthologue Optix is an essential regulator of neuroepithelial maintenance and patterning in the Drosophila brain. Six3 and Six6 are required for mammalian eye and forebrain development, and mutations in humans are associated with severe eye and brain malformation. In Drosophila, Optix is expressed in a sharply defined region of the larval optic lobe, and its expression is reciprocal to that of the transcription factor Vsx1. Optix gain- and loss-of-function affects neuroepithelial adhesion, integrity and polarity. Restricted cell lineage boundaries were found that correspond to transcription factor expression domains. It is proposed that the optic lobe is compartmentalised by expression of Optix and Vsx1. These findings provide insight into the spatial patterning of a complex region of the brain, and suggest an evolutionarily conserved principle of visual system development.
Laranjeiro, R. and Whitmore, D. (2014). Transcription factors involved in retinogenesis are co-opted by the circadian clock following photoreceptor differentiation. Development 141: 2644-2656. PubMed ID: 24924194
The circadian clock is known to regulate a wide range of physiological and cellular processes, yet remarkably little is known about its role during embryo development. Zebrafish offer a unique opportunity to explore this issue, not only because a great deal is known about key developmental events in this species, but also because the clock starts on the very first day of development. This study identified numerous rhythmic genes in zebrafish larvae, including the key transcriptional regulators neurod (Drosophila homolog: Tap) and cdx1b (Drosophila homolog: Caudal), which are involved in neuronal and intestinal differentiation, respectively. Rhythmic expression of neurod and several additional transcription factors was only observed in the developing retina. Surprisingly, these rhythms in expression commenced at a stage of development after these transcription factors are known to have played their essential role in photoreceptor differentiation. Furthermore, this circadian regulation was maintained in adult retina. Thus, once mature photoreceptors are formed, multiple retinal transcription factors fall under circadian clock control, at which point they appear to play a new and important role in regulating rhythmic elements in the phototransduction pathway.
Ebert, A. M., Childs, S. J., Hehr, C. L., Cechmanek, P. B. and McFarlane, S. (2014). Sema6a and Plxna2 mediate spatially regulated repulsion within the developing eye to promote eye vesicle cohesion. Development 141: 2473-2482. PubMed ID: 24917502
Organs are generated from collections of cells that coalesce and remain together as they undergo a series of choreographed movements to give the organ its final shape. Little is known about the cellular and molecular mechanisms that regulate tissue cohesion during morphogenesis. Extensive cell movements underlie eye development, starting with the eye field separating to form bilateral vesicles that go on to evaginate from the forebrain. What keeps eye cells together as they undergo morphogenesis and extensive proliferation is unknown. This study shows that plexina2 (Plxna2; see Drosophila Plexin A), a member of a receptor family best known for its roles in axon and cell guidance, is required alongside the repellent semaphorin 6a to keep cells integrated within the zebrafish eye vesicle epithelium. sema6a is expressed throughout the eye vesicle, whereas plxna2 is restricted to the ventral vesicle. Knockdown of Plxna2 or Sema6a results in a loss of vesicle integrity, with time-lapse microscopy showing that eye progenitors either fail to enter the evaginating vesicles or delaminate from the eye epithelium. Explant experiments, and rescue of eye vesicle integrity with simultaneous knockdown of sema6a and plxna2, point to an eye-autonomous requirement for Sema6a/Plxna2. A novel, tissue-autonomous mechanism of organ cohesion is proposed, with neutralization of repulsion suggested as a means to promote interactions between cells within a tissue domain.
Dutta, D. J., Zameer, A., Mariani, J. N., Zhang, J., Asp, L., Huynh, J., Mahase, S., Laitman, B. M., Argaw, A. T., Mitiku, N., Urbanski, M., Melendez-Vasquez, C. V., Casaccia, P., Hayot, F., Bottinger, E. P., Brown, C. W. and John, G. R. (2014). Combinatorial actions of Tgfβ and Activin ligands promote oligodendrocyte development and CNS myelination. Development 141: 2414-2428. PubMed ID: 24917498
In the embryonic CNS, development of myelin-forming oligodendrocytes is limited by bone morphogenetic proteins, which constitute one arm of the transforming growth factor-beta (Tgfβ) family and signal canonically via Smads 1/5/8. Tgfβ ligands and Activins comprise the other arm and signal via Smads 2/3, but their roles in oligodendrocyte development are incompletely characterized. This study reports that Tgfβ ligands and activin B (ActB; see Drosophila Activin) act in concert in the mammalian spinal cord to promote oligodendrocyte generation and myelination. In mouse neural tube, newly specified oligodendrocyte progenitors (OLPs) are first exposed to Tgfβ ligands in isolation, then later in combination with ActB during maturation. In primary OLP cultures, Tgfβ1 and ActB differentially activate canonical Smad3 (see Drosophila Smox) and non-canonical MAP kinase signaling. Both ligands enhance viability, and Tgfβ1 promotes proliferation while ActB supports maturation. Importantly, co-treatment strongly activates both signaling pathways, producing an additive effect on viability and enhancing both proliferation and differentiation such that mature oligodendrocyte numbers are substantially increased. Co-treatment promotes myelination in OLP-neuron co-cultures, and maturing oligodendrocytes in spinal cord white matter display strong Smad3 and MAP kinase activation. In spinal cords of ActB-deficient inhibin Inhbb-/- embryos, apoptosis in the oligodendrocyte lineage is increased and OLP numbers transiently reduced, but numbers, maturation and myelination recover during the first postnatal week. Smad3-/- mice display a more severe phenotype, including diminished viability and proliferation, persistently reduced mature and immature cell numbers, and delayed myelination. Collectively, these findings suggest that, in mammalian spinal cord, Tgfβ ligands and ActB together support oligodendrocyte development and myelin formation.
Wednesday, August 6th
Korenjak, M., Kwon, E., Morris, R. T., Anderssen, E., Amzallag, A., Ramaswamy, S., Dyson, N. J. (2014). dREAM co-operates with insulator-binding proteins and regulates expression at divergently paired genes. Nucleic Acids Res. PubMed ID: 25053843
dREAM (Drosophila Rb E2F and Myb) complexes represent the predominant form of E2F/RBF repressor complexes in Drosophila. dREAM associates with thousands of sites in the fly genome but its mechanism of action is unknown. To understand the genomic context in which dREAM acts, the distribution and localization of Drosophila E2F and dREAM proteins were examined. This study reports a striking and unexpected overlap between dE2F2/dREAM sites and binding sites for the insulator-binding proteins CP190 and Beaf-32. Genetic assays show that these components functionally co-operate and chromatin immunoprecipitation experiments on mutant animals demonstrate that dE2F2 is important for association of CP190 with chromatin. dE2F2/dREAM binding sites are enriched at divergently transcribed genes, and the majority of genes upregulated by dE2F2 depletion represent the repressed half of a differentially expressed, divergently transcribed pair of genes. Analysis of mutant animals confirms that dREAM and CP190 are similarly required for transcriptional integrity at these gene pairs and suggest that dREAM functions in concert with CP190 to establish boundaries between repressed/activated genes. Consistent with the idea that dREAM co-operates with insulator-binding proteins, genomic regions bound by dREAM possess enhancer-blocking activity that depends on multiple dREAM components. These findings suggest that dREAM functions in the organization of transcriptional domains.
Gonzalez, I., Mateos-Langerak, J., Thomas, A., Cheutin, T. and Cavalli, G. (2014). Identification of regulators of the three-dimensional polycomb organization by a microscopy-based genome-wide RNAi screen. Mol Cell 54: 485-499. PubMed ID: 24703951
Polycomb group (PcG) proteins dynamically define cellular identities through epigenetic repression of key developmental genes. PcG target gene repression can be stabilized through the interaction in the nucleus at PcG foci. This study report the results of a high-resolution microscopy genome-wide RNAi screen that identifies 129 genes that regulate the nuclear organization of Pc foci. Candidate genes include PcG components and chromatin factors, as well as many protein-modifying enzymes, including components of the SUMOylation pathway. In the absence of SUMO, Pc foci coagulate into larger aggregates. Conversely, loss of function of the SUMO peptidase Velo disperses Pc foci. Moreover, SUMO and Velo colocalize with PcG proteins at PREs, and Pc SUMOylation affects its chromatin targeting, suggesting that the dynamic regulation of Pc SUMOylation regulates PcG-mediated silencing by modulating the kinetics of Pc binding to chromatin as well as its ability to form Polycomb foci.
Zhang, C., Tinto, S. C., Li, G., Lin, N., Chung, M., Moreno, E., Moberg, K. H. and Zhou, L. (2014). An intergenic regulatory region mediates Drosophila Myc-induced apoptosis and blocks tissue hyperplasia. Oncogene [Epub ahead of print]. PubMed ID: 24931167
Induction of cell-autonomous apoptosis following oncogene-induced overproliferation is a major tumor-suppressive mechanism in vertebrates. However, the detailed mechanism mediating this process remains enigmatic. This study demonstrates that dMyc-induced cell-autonomous apoptosis in the fruit fly Drosophila relies on an intergenic sequence termed the IRER (irradiation-responsive enhancer region). The IRER mediates the expression of surrounding proapoptotic genes, and an in vivo reporter of the IRER chromatin state was used to gather evidence that epigenetic control of DNA accessibility within the IRER is an important determinant of the strength of this response to excess dMyc. In a previous work, it was shown that the IRER also mediates P53-dependent induction of proapoptotic genes following DNA damage, and the chromatin conformation within IRER is regulated by polycomb group-mediated histone modifications. dMyc-induced apoptosis and the P53-mediated DNA damage response thus overlap in a requirement for the IRER. The epigenetic mechanisms controlling IRER accessibility appear to set thresholds for the P53- and dMyc-induced expression of apoptotic genes in vivo and may have a profound impact on cellular sensitivity to oncogene-induced stress.
Drewell, R. A., Bush, E. C., Remnant, E. J., Wong, G. T., Beeler, S. M., Stringham, J. L., Lim, J. and Oldroyd, B. P. (2014). The dynamic DNA methylation cycle from egg to sperm in the honey bee Apis mellifera. Development 141: 2702-2711. PubMed ID: 24924193
In honey bees (Apis mellifera), the epigenetic mark of DNA methylation is central to the developmental regulation of caste differentiation, but may also be involved in additional biological functions. This study examined the whole genome methylation profiles of three stages of the haploid honey bee genome: unfertilised eggs, the adult drones that develop from these eggs and the sperm produced by these drones. These methylomes reveal distinct patterns of methylation. Eggs and sperm show 381 genes with significantly different CpG methylation patterns, with the vast majority being more methylated in eggs. Adult drones show greatly reduced levels of methylation across the genome when compared with both gamete samples. This suggests a dynamic cycle of methylation loss and gain through the development of the drone and during spermatogenesis. Although fluxes in methylation during embryogenesis may account for some of the differentially methylated sites, the distinct methylation patterns at some genes suggest parent-specific epigenetic marking in the gametes. Extensive germ line methylation of some genes possibly explains the lower-than-expected frequency of CpG sites in these genes. The potential developmental and evolutionary implications are discussed of methylation in eggs and sperm in this eusocial insect species.
Tuesday, August 5th
Galili, D. S., Dylla, K. V., Ludke, A., Friedrich, A. B., Yamagata, N., Wong, J. Y., Ho, C. H., Szyszka, P. and Tanimoto, H. (2014). Converging Circuits Mediate Temperature and Shock Aversive Olfactory Conditioning in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 25042591
Drosophila learn to avoid odors that are paired with aversive stimuli. Electric shock is a potent aversive stimulus that acts via dopamine neurons to elicit avoidance of the associated odor. While dopamine signaling has been demonstrated to mediate olfactory electric shock conditioning, it remains unclear how this pathway is involved in other types of behavioral reinforcement, such as in learned avoidance of odors paired with increased temperature. To better understand the neural mechanisms of distinct aversive reinforcement signals, an olfactory temperature conditioning assay were established comparable to olfactory electric shock conditioning. The AC neurons, which are internal thermal receptors expressing dTrpA1, are selectively required for odor-temperature but not for odor-shock memory. Furthermore, these separate sensory pathways for increased temperature and shock converge onto overlapping populations of dopamine neurons that signal aversive reinforcement. Temperature conditioning appears to require a subset of the dopamine neurons required for electric shock conditioning. It is concluded that dopamine neurons integrate different noxious signals into a general aversive reinforcement pathway.
Behnia, R., Clark, D. A., Carter, A. G., Clandinin, T. R., Desplan, C. (2014). Processing properties of ON and OFF pathways for Drosophila motion detection. Nature [Epub ahead of print]. PubMed ID: 25043016
The algorithms and neural circuits that process spatio-temporal changes in luminance to extract visual motion cues have been the focus of intense research. An influential model, the Hassenstein-Reichardt correlator, relies on differential temporal filtering of two spatially separated input channels, delaying one input signal with respect to the other. Motion in a particular direction causes these delayed and non-delayed luminance signals to arrive simultaneously at a subsequent processing step in the brain; these signals are then nonlinearly amplified to produce a direction-selective response. Recent work in Drosophila has identified two parallel pathways that selectively respond to either moving light or dark edges. Each of these pathways requires two critical processing steps to be applied to incoming signals: differential delay between the spatial input channels, and distinct processing of brightness increment and decrement signals. This study demonstrates, using in vivo patch-clamp recordings, that four medulla neurons implement these two processing steps. The neurons Mi1 and Tm3 respond selectively to brightness increments, with the response of Mi1 delayed relative to Tm3. Conversely, Tm1 and Tm2 respond selectively to brightness decrements, with the response of Tm1 delayed compared with Tm2. Remarkably, constraining Hassenstein–Reichardt correlator models using these measurements produces outputs consistent with previously measured properties of motion detectors, including temporal frequency tuning and specificity for light versus dark edges. It is proposed that Mi1 and Tm3 perform critical processing of the delayed and non-delayed input channels of the correlator responsible for the detection of light edges, while Tm1 and Tm2 play analogous roles in the detection of moving dark edges. The data show that specific medulla neurons possess response properties that allow them to implement the algorithmic steps that precede the correlative operation in the Hassenstein–Reichardt correlator, revealing elements of the long-sought neural substrates of motion detection in the fly.
Das, G., Klappenbach, M., Vrontou, E., Perisse, E., Clark, C. M., Burke, C. J. and Waddell, S. (2014). Drosophila learn opposing components of a compound food stimulus. Curr Biol [Epub ahead of print]. PubMed ID: 25042590
Dopaminergic neurons provide value signals in mammals and insects. During Drosophila olfactory learning, distinct subsets of dopaminergic neurons appear to assign either positive or negative value to odor representations in mushroom body neurons. However, it is not known how flies evaluate substances that have mixed valence. This study shows that flies form short-lived aversive olfactory memories when trained with odors and sugars that are contaminated with the common insect repellent DEET. This DEET-aversive learning required the MB-MP1 dopaminergic neurons that are also required for shock learning. Moreover, differential conditioning with DEET versus shock suggests that formation of these distinct aversive olfactory memories relies on a common negatively reinforcing dopaminergic mechanism. Surprisingly, as time passed after training, the behavior of DEET-sugar-trained flies reversed from conditioned odor avoidance into odor approach. In addition, flies that were compromised for reward learning exhibited a more robust and longer-lived aversive-DEET memory. These data demonstrate that flies independently process the DEET and sugar components to form parallel aversive and appetitive olfactory memories, with distinct kinetics, that compete to guide learned behavior.
Hanus, C., Kochen, L., Tom Dieck, S., Racine, V., Sibarita, J. B., Schuman, E. M. and Ehlers, M. D. (2014). Synaptic control of secretory trafficking in dendrites. Cell Rep 7: 1771-1778. PubMed ID: 24931613
Localized signaling in neuronal dendrites requires tight spatial control of membrane composition. Upon initial synthesis, nascent secretory cargo in dendrites exits the endoplasmic reticulum (ER) from local zones of ER complexity that are spatially coupled to post-ER compartments. Although newly synthesized membrane proteins can be processed locally, the mechanisms that control the spatial range of secretory cargo transport in dendritic segments are unknown. This study, carried out in neuronal cell cultures, monitored the dynamics of nascent membrane proteins in dendritic post-ER compartments under regimes of low or increased neuronal activity. In response to activity blockade, post-ER carriers are highly mobile and are transported over long distances. Conversely, increasing synaptic activity dramatically restricts the spatial scale of post-ER trafficking along dendrites. This activity-induced confinement of secretory cargo requires site-specific phosphorylation of the kinesin motor Kif17 (see Drosophila KIF17) by Ca2+/calmodulin-dependent protein kinases (CaMK) (see for example Drosophila CaMKII). Thus, the length scales of early secretory trafficking in dendrites are tuned by activity-dependent regulation of microtubule-dependent transport.
Monday, August 4th
Jagadish, S., Barnea, G., Clandinin, T. R., Axel, R. (2014). Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace. Neuron. PubMed ID: 25043419
In Drosophila, the four inner photoreceptor neurons
exhibit overlapping but distinct spectral sensitivities
and mediate behaviors that reflect spectral preference.
A genetic strategy, Tango-Trace, permits the identification of the
connections of the four chromatic photoreceptors.
Each of the four stochastically distributed chromatic
photoreceptor subtypes make distinct connections
in the medulla with four different TmY cells. Moreover,
each class of TmY cells forms a retinotopic
map in both the medulla and the lobula complex,
generating four overlapping topographic maps that
could carry different color information. Thus, the four inner photoreceptors transmit spectral information through distinct channels that may converge in
both the medulla and lobula complex. These projections
could provide an anatomic basis for color vision
and may relay information about color to motion sensitive
areas. Moreover, the Tango-Trace strategy may be applied more generally to identify neural circuits in the fly brain.
Laranjeiro, R. and Whitmore, D. (2014). Transcription factors involved in retinogenesis are co-opted by the circadian clock following photoreceptor differentiation. Development 141: 2644-2656. PubMed ID: 24924194
The circadian clock is known to regulate a wide range of physiological and cellular processes, yet remarkably little is known about its role during embryo development. Zebrafish offer a unique opportunity to explore this issue, not only because a great deal is known about key developmental events in this species, but also because the clock starts on the very first day of development. This study identified numerous rhythmic genes in zebrafish larvae, including the key transcriptional regulators neurod (homolog of Drosophila Target of Pox-n) and cdx1b (homolog of Drosophila Caudal), which are involved in neuronal and intestinal differentiation, respectively. Rhythmic expression of neurod and several additional transcription factors was only observed in the developing retina. Surprisingly, these rhythms in expression commenced at a stage of development after these transcription factors are known to have played their essential role in photoreceptor differentiation. Furthermore, this circadian regulation was maintained in adult retina. Thus, once mature photoreceptors are formed, multiple retinal transcription factors fall under circadian clock control, at which point they appear to play a new and important role in regulating rhythmic elements in the phototransduction pathway.
Zakaria, S., Mao, Y., Kuta, A., Ferreira de Sousa, C., Gaufo, G. O., McNeill, H., Hindges, R., Guthrie, S., Irvine, K. D. and Francis-West, P. H. (2014). Regulation of neuronal migration by Dchs1-Fat4 planar cell polarity. Curr Biol 24(14): 1620-7. PubMed ID: 24998526
Planar cell polarity (PCP) describes the polarization of cell structures and behaviors within the plane of a tissue. PCP is essential for the generation of tissue architecture during embryogenesis and for postnatal growth and tissue repair, yet how it is oriented to coordinate cell polarity remains poorly understood. In Drosophila, PCP is mediated via the Frizzled-Flamingo (Fz-PCP) and Dachsous-Fat (Fat-PCP) pathways. Fz-PCP is conserved in vertebrates, but an understanding in vertebrates of whether and how Fat-PCP polarizes cells, and its relationship to Fz-PCP signaling, is lacking. Mutations in human FAT4 (see Drosophila Fat) and DCHS1 (see Drosophila Dachsous), key components of Fat-PCP signaling, cause Van Maldergem syndrome, characterized by severe neuronal abnormalities indicative of altered neuronal migration. This study investigated the role and mechanisms of Fat-PCP during neuronal migration using the murine facial branchiomotor (FBM) neurons as a model. Fat4 and Dchs1 were found to be expressed in complementary gradients and are required for the collective tangential migration of FBM neurons and for their PCP. Fat4 and Dchs1 are required intrinsically within the FBM neurons and extrinsically within the neuroepithelium. Remarkably, Fat-PCP and Fz-PCP regulate FBM neuron migration along orthogonal axes. Disruption of the Dchs1 gradients by mosaic inactivation of Dchs1 alters FBM neuron polarity and migration. This study implies that PCP in vertebrates can be regulated via gradients of Fat4 and Dchs1 expression, which establish intracellular polarity across FBM cells during their migration. The results also identify Fat-PCP as a novel neuronal guidance system and reveal that Fat-PCP and Fz-PCP can act along orthogonal axes.
Caronia-Brown, G., Yoshida, M., Gulden, F., Assimacopoulos, S. and Grove, E. A. (2014). The cortical hem regulates the size and patterning of neocortex. Development 141: 2855-2865. PubMed ID: 24948604
The cortical hem, a source of Wingless-related (WNT) and bone morphogenetic protein (BMP) signaling in the dorsomedial telencephalon, is the embryonic organizer for the hippocampus. Whether the hem is a major regulator of cortical patterning outside the hippocampus has not been investigated. This study examined regional organization across the entire cerebral cortex in mice genetically engineered to lack the hem. Indicating that the hem regulates dorsoventral patterning in the cortical hemisphere, the neocortex, particularly dorsomedial neocortex, was reduced in size in late-stage hem-ablated embryos, whereas cortex ventrolateral to the neocortex expanded dorsally. Unexpectedly, hem ablation also perturbed regional patterning along the rostrocaudal axis of neocortex. Rostral neocortical domains identified by characteristic gene expression were expanded, and caudal domains diminished. A similar shift occurs when fibroblast growth factor (FGF) 8 (see Drosophila Pyramus and Thisbe) is increased at the rostral telencephalic organizer, yet the FGF8 source was unchanged in hem-ablated brains. Rather hem WNT or BMP signals, or both, were found to have opposite effects to those of FGF8 in regulating transcription factors that control the size and position of neocortical areas. When the hem is ablated a necessary balance is perturbed, and cerebral cortex is rostralized. These findings reveal a much broader role for the hem in cortical development than previously recognized, and emphasize that two major signaling centers interact antagonistically to pattern cerebral cortex.
Sunday, August 3rd
Borromeo, M. D., Meredith, D. M., Castro, D. S., Chang, J. C., Tung, K. C., Guillemot, F. and Johnson, J. E. (2014). A transcription factor network specifying inhibitory versus excitatory neurons in the dorsal spinal cord. Development 141: 2803-2812. PubMed ID: 24924197
The proper balance of excitatory and inhibitory neurons is crucial for normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors (TFs), Ascl1 and Ptf1a, have contrasting functions in specifying these neurons. To understand how Ascl1 and Ptf1a function in this process, their direct transcriptional targets were tested genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. Ascl1 and Ptf1a were shown to directly regulate distinct homeodomain TFs that specify excitatory or inhibitory neuronal fates. In addition, Ascl1 directly regulates genes with roles in several steps of the neurogenic program, including Notch signaling, neuronal differentiation, axon guidance and synapse formation. By contrast, Ptf1a directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, Ptf1a represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Ascl1 and Ptf1a bind sequences primarily enriched for a specific E-Box motif (CAGCTG) and for secondary motifs used by Sox, Rfx, Pou and homeodomain factors. Ptf1a also binds sequences uniquely enriched in the CAGATG E-box and in the binding motif for its co-factor Rbpj (see Drosophila Suppressor of Hairless), providing two factors that influence the specificity of Ptf1a binding. The direct transcriptional targets identified for Ascl1 and Ptf1a provide a molecular understanding of how these DNA-binding proteins function in neuronal development, particularly as key regulators of homeodomain TFs required for neuronal subtype specification.
Ali, F. R., Cheng, K., Kirwan, P., Metcalfe, S., Livesey, F. J., Barker, R. A. and Philpott, A. (2014). The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro. Development 141: 2216-2224. PubMed ID: 24821983
Generation of neurons from patient fibroblasts using a combination of developmentally defined transcription factors has great potential in disease modelling, as well as ultimately for use in regeneration and repair. However, generation of physiologically mature neurons in vitro remains problematic. This study demonstrates the cell-cycle-dependent phosphorylation of a key reprogramming transcription factor, Ascl1, on multiple serine-proline sites. This multisite phosphorylation is a crucial regulator of the ability of Ascl1 to drive neuronal differentiation and maturation in vivo in the developing embryo; a phosphomutant form of Ascl1 shows substantially enhanced neuronal induction activity in Xenopus embryos. Mechanistically, this un(der)phosphorylated Ascl1 is resistant to inhibition by both cyclin-dependent kinase activity and Notch signalling, both of which normally limit its neurogenic potential. Ascl1 is a central component of reprogramming transcription factor cocktails to generate neurons from human fibroblasts; the use of phosphomutant Ascl1 in place of the wild-type protein significantly promotes neuronal maturity after human fibroblast reprogramming in vitro. These results demonstrate that cell-cycle-dependent post-translational modification of proneural proteins directly regulates neuronal differentiation in vivo during development, and that this regulatory mechanism can be harnessed to promote maturation of neurons obtained by transdifferentiation of human cells in vitro.
Copf, T. (2014). Developmental shaping of dendritic arbors in Drosophila relies on tightly regulated intra-neuronal activity of protein kinase A (PKA). Dev Biol [Epub ahead of print]. PubMed ID: 25017992
Dendrites develop morphologies characterized by multiple levels of complexity that involve neuron type specific dendritic length and particular spatial distribution. How this is developmentally regulated and in particular which signaling molecules are crucial in the process is still not understood. Using Drosophila class IV dendritic arborization (da) neurons this study tested in vivo the effects of cell-autonomous dose-dependent changes in the activity levels of the cAMP-dependent Protein Kinase A (PKA) on the formation of complex dendritic arbors. Genetic manipulations of the PKA activity levels were found to affect profoundly the arbor complexity with strongest impact on distal branches. Both decreasing and increasing PKA activity result in a reduced complexity of the arbors, as reflected in decreased dendritic length and number of branching points, suggesting an inverted U-shape response to PKA. The phenotypes are accompanied by changes in organelle distribution: Golgi outpost and early endosomes in distal dendritic branches are reduced in PKA mutants. By using Rab5 dominant negative it was found that PKA interacts genetically with the early endosomal pathway. Tests were performed of whether the possible relationship between PKA and organelles may be the result of phosphorylation of the microtubule motor dynein components or Rab5. Cytoplasmic dynein components were found to be direct PKA phosphorylation targets in vitro, but not in vivo, thus pointing to a different putative in vivo target. The data argue that tightly controlled dose-dependent intra-neuronal PKA activity levels are critical in determining the dendritic arbor complexity, one of the possible ways being through the regulation of organelle distribution.
Shklyar, B., Sellman, Y., Shklover, J., Mishnaevski, K., Levy-Adam, F. and Kurant, E. (2014). Developmental regulation of glial cell phagocytic function during Drosophila embryogenesis. Dev Biol. PubMed ID: 25046770
The proper removal of superfluous neurons through apoptosis and subsequent phagocytosis is essential for normal development of the central nervous system (CNS). During Drosophila embryogenesis, a large number of apoptotic neurons are efficiently engulfed and degraded by phagocytic glia. This study demonstrates that glial proficiency to phagocytose relies on expression of phagocytic receptors for apoptotic cells, Simu (Nimrod C4) and Drpr (Draper). Moreover, the phagocytic ability of embryonic glia was shown to be established as part of a developmental program responsible for glial cell fate determination and is not triggered by apoptosis per se. Explicitly, evidence is provided for a critical role of the major regulators of glial identity, gcm and repo, in controlling glial phagocytic function through regulation of Simu and Drpr specific expression. Taken together, this study uncovers molecular mechanisms essential for establishment of embryonic glia as primary phagocytes during CNS development.
Saturday, August 2nd
Herren, J. K., Paredes, J. C., Schupfer, F., Arafah, K., Bulet, P. and Lemaitre, B. (2014). Insect endosymbiont proliferation is limited by lipid availability. Elife: e02964. PubMed ID: 25027439
Spiroplasma poulsonii is a maternally transmitted bacterial endosymbiont that is naturally associated with Drosophila melanogaster. S. poulsonii resides extracellularly in the hemolymph, where it must acquire metabolites to sustain proliferation. This study found that Spiroplasma proliferation specifically depletes host hemolymph diacylglyceride, the major lipid class transported by the lipoprotein, Lpp. RNAi-mediated knockdown of Lpp expression, which reduces the amount of circulating lipids, inhibits Spiroplasma proliferation demonstrating that bacterial proliferation requires hemolymph-lipids. Altogether, this study shows that an insect endosymbiont acquires specific lipidic metabolites from the transport lipoproteins in the hemolymph of its host. In addition, this study showed that the proliferation of this endosymbiont is limited by the availability of hemolymph lipids. This feature could limit endosymbiont over-proliferation under conditions of host nutrient limitation as lipid availability is strongly influenced by the nutritional state.
Choi, J. Y. and Aquadro, C. F. (2014). The coevolutionary period of Wolbachia pipientis infecting Drosophila ananassae and its impact on the evolution of the host germline stem cell regulating genes. Mol Biol Evol. PubMed ID: 24974378
The endosymbiotic bacteria Wolbachia pipientis is known to infect a wide range of arthropod species yet less is known about the coevolutionary history it has with its hosts. Evidence of highly identical W. pipientis strains in evolutionary divergent hosts suggests horizontal transfer between hosts. For example, Drosophila ananassae is infected with a W. pipientis strain that is nearly identical in sequence to a strain that infects both D. simulans and D. suzukii, suggesting recent horizontal transfer among these three species. However, it is unknown whether the W. pipientis strain had recently invaded all three species or a more complex infectious dynamic underlies the horizontal transfers. This study examined the coevolutionary history of D. ananassae and its resident W. pipientis to infer its period of infection. Phylogenetic analysis of D. ananassae mitochondrial DNA and W. pipientis DNA sequence diversity revealed the current W. pipientis infection is not recent. In addition, the population genetics and molecular evolution of several Germline Stem Cell (GSC) regulating genes of D. ananassae were examined. These studies reveal significant evidence of recent and long-term positive selection at stonewall in D. ananassae, while pumillio showed patterns of variation consistent with only recent positive selection. Previous studies had found evidence for adaptive evolution of two key germline differentiation genes, bag of marbles (bam) and benign gonial cell neoplasm (bgcn), in D. melanogaster and D. simulans, and it was proposed that the adaptive evolution at these two genes was driven by arms race between the host GSC and W. pipientis. However, this study did not find any statistical departures from a neutral model of evolution for bam and bgcn in D. ananassae despite the new evidence that this species has been infected with W. pipientis for a period longer than the most recent infection in D. melanogaster. In the end analyzing the GSC regulating genes individually showed two out of the seven genes to have evidence of selection. However, combining the dataset and fitting a specific population genetic model, significant proportion of the nonsynonymous sites across the GSC regulating genes were driven to fixation by positive selection. Clearly the GSC system is under rapid evolution and potentially multiple drivers are causing the rapid evolution.
Engel, E., Viargues, P., Mortier, M., Taillebourg, E., Coute, Y., Thevenon, D. and Fauvarque, M. O. (2014). Identifying USPs regulating immune signals in Drosophila:
USP2 deubiquitinates Imd and promotes its degradation by interacting with the proteasome. Cell Commun Signal 12: 41. PubMed ID: 25027767
Rapid activation of innate immune defences upon microbial infection depends on the evolutionary conserved NFκB-dependent signals, which deregulation is frequently associated with chronic inflammation and oncogenesis. These signals are tightly regulated by the linkage of different kinds of ubiquitin moieties on proteins that modify either their activity or their stability. To investigate how ubiquitin specific proteases (USPs) orchestrate immune signal regulation, a focused RNA interference library was created and screened on Drosophila NFκB-like pathways Toll and Imd in cultured S2 cells, and the function of selected genes were further analysed in vivo. USP2 and USP34/Puf, in addition to the previously described USP36/Scny, prevent inappropriate activation of Imd-dependent immune signal in unchallenged conditions. Moreover, USP34 is also necessary to prevent constitutive activation of the Toll pathway. However, while USP2 also prevents excessive Imd-dependent signalling in vivo, USP34 shows differential requirement depending on NFκB target genes, in response to fly infection by either Gram-positive or Gram-negative bacteria. It was further shown that USP2 prevents the constitutive activation of signalling by promoting Imd proteasomal degradation. Indeed, the homeostasis of the Imd scaffolding molecule is tightly regulated by the linkage of lysine 48-linked ubiquitin chains (K48) acting as a tag for its proteasomal degradation. This process is necessary to prevent constitutive activation of Imd pathway in vivo and is inhibited in response to infection. The control of Imd homeostasis by USP2 is associated with the hydrolysis of Imd linked K48-ubiquitin chains and the synergistic binding of USP2 and Imd to the proteasome, as evidenced by both mass-spectrometry analysis of USP2 partners and by co-immunoprecipitation experiments. This work identified one known (USP36) and two new (USP2, USP34) ubiquitin specific proteases regulating Imd or Toll dependent immune signalling in Drosophila. It further highlights the ubiquitin dependent control of Imd homeostasis and shows a new activity for USP2 at the proteasome allowing for Imd degradation. This study provides original information for the better understanding of the strong implication of USP2 in pathological processes in humans, including cancerogenesis.
Tan, K. L., Vlisidou, I. and Wood, W. (2014). Ecdysone mediates the development of immunity in the Drosophila embryo. Curr Biol 24: 1145-1152. PubMed ID: 24794300
Beyond their role in cell metabolism, development, and reproduction, hormones are also important modulators of the immune system. In the context of inflammatory disorders, systemic administration of pharmacological doses of synthetic glucocorticoids (GCs) is widely used as an anti-inflammatory treatment. However, not all actions of GCs are immunosuppressive, and many studies have suggested that physiological concentrations of GCs can have immunoenhancing effects. For a more comprehensive understanding of how steroid hormones regulate immunity and inflammation, a simple in vivo system is required. The Drosophila embryo has recently emerged as a powerful model system to study the recruitment of immune cells to sterile wounds and host-pathogen dynamics. This study investigated the immune response of the fly embryo to bacterial infections and found that the steroid hormone 20-hydroxyecdysone (20-HE) can regulate the quality of the immune response and influence the resolution of infection in Drosophila embryos.
Friday, August 1st
Ghavi-Helm, Y., Klein, F. A., Pakozdi, T., Ciglar, L., Noordermeer, D., Huber, W., Furlong, E. E. (2014). Enhancer loops appear stable during development and are associated with paused polymerase. Nature [Epub ahead of print]. PubMed ID: 25043061
Developmental enhancers initiate transcription and are fundamental to understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed. The extent to which preformed enhancer-promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. This study generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. These results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer-promoter loops through release of paused polymerase.
Zehavi, Y., Sloutskin, A., Kuznetsov, O. and Juven-Gershon, T. (2014). The core promoter composition establishes a new dimension in developmental gene networks. Nucleus 5:4, 1-6. PubMed ID: 25032831
Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II, which initiates transcription at the core promoter. The dorsal-ventral gene regulatory network (GRN) includes multiple genes that are activated by different nuclear concentrations of the Dorsal transcription factor along the dorsal-ventral axis. Downstream core promoter element (DPE)-containing genes are conserved and highly prevalent among Dorsal target genes. Moreover, the DPE motif is functional in multiple Dorsal target genes, as mutation of the DPE results in the loss of transcriptional activity. Furthermore, analysis of hybrid enhancer-promoter constructs reveals that the core promoter composition plays a pivotal role in the transcriptional output. Importantly, in vivo evidence is provided that expression driven by the homeotic Antennapedia P2 promoter during Drosophila embryogenesis is dependent on the DPE. Taken together, it is proposed that transcriptional regulation results from the interplay between enhancers and core promoter composition, thus establishing a novel dimension in developmental GRNs.
Nakamura, R., Tsukahara, T., Qu, W., Ichikawa, K., Otsuka, T., Ogoshi, K., Saito, T. L., Matsushima, K., Sugano, S., Hashimoto, S., Suzuki, Y., Morishita, S. and Takeda, H. (2014). Large hypomethylated domains serve as strong repressive machinery for key developmental genes in vertebrates. Development 141: 2568-2580. PubMed ID: 24924192
DNA methylation is a fundamental epigenetic modification in vertebrate genomes and a small fraction of genomic regions is hypomethylated. Previous studies have implicated hypomethylated regions in gene regulation, but their functions in vertebrate development remain elusive. To address this issue, epigenomic profiles were generated that include base-resolution DNA methylomes and histone modification maps from both pluripotent cells and mature organs of medaka fish, and the profiles were compared with those of human ES cells. It was found that a subset of hypomethylated domains harbor H3K27me3 (K27HMDs) and their size positively correlates with the accumulation of H3K27me3. Large K27HMDs are conserved between medaka and human pluripotent cells and predominantly contain promoters of developmental transcription factor genes. These key genes were found to be under strong transcriptional repression, when compared with other developmental genes with smaller K27HMDs. Furthermore, human-specific K27HMDs show an enrichment of neuronal activity-related genes, which suggests a distinct regulation of these genes in medaka and human. In mature organs, some of the large HMDs become shortened by elevated DNA methylation and associate with sustained gene expression. This study highlights the significance of domain size in epigenetic gene regulation. It is proposed that large K27HMDs play a crucial role in pluripotent cells by strictly repressing key developmental genes, whereas their shortening consolidates long-term gene expression in adult differentiated cells.
Brittain, A., Stroebele, E. and Erives, A. (2014). Microsatellite repeat instability fuels evolution of embryonic enhancers in hawaiian Drosophila. PLoS One 9: e101177. PubMed ID: 24978198
For approximately 30 million years, the eggs of Hawaiian Drosophila were laid in ever-changing environments caused by high rates of island formation. The associated diversification of the size and developmental rate of the syncytial fly embryo would have altered morphogenic gradients, thus necessitating frequent evolutionary compensation of transcriptional responses. The consequences these radiations had on transcriptional enhancers patterning the embryo were investigated to see whether their pattern of molecular evolution is different from non-Hawaiian species. The Neurogenic Ectoderm Enhancers from two different Hawaiian Drosophila groups were identified and functionally assayed in transgenic D. melanogaster: (1) the picture wing group, and (2) the modified mouthparts group. The binding sites in this set of well-characterized enhancers were found to be footprinted by diverse microsatellite repeat (MSR) sequences. It was further shown that Hawaiian embryonic enhancers in general are enriched in MSR relative to both Hawaiian non-embryonic enhancers and non-Hawaiian embryonic enhancers. It is proposed that embryonic enhancers are sensitive to Activator spacing because they often serve as assembly scaffolds for the aggregation of transcription factor activator complexes. Furthermore, as most indels are produced by microsatellite repeat slippage, enhancers from Hawaiian Drosophila lineages, which experience dynamic evolutionary pressures, would become grossly enriched in MSR content.
Senecal, A., Munsky, B., Proux, F., Ly, N., Braye, F. E., Zimmer, C., Mueller, F. and Darzacq, X. (2014). Transcription factors modulate c-Fos transcriptional bursts. Cell Rep 8(1):75-83. PubMed ID: 24981864
Transcription is a stochastic process occurring mostly in episodic bursts. Although the local chromatin environment is known to influence the bursting behavior on long timescales, the impact of transcription factors (TFs)-especially in rapidly inducible systems-is largely unknown. Using fluorescence in situ hybridization and computational models, this study quantified the transcriptional activity of the proto-oncogene c-Fos (see Drosophila Fos) with single mRNA accuracy at individual endogenous alleles. It was shown that, during MAPK induction, the TF concentration modulates the burst frequency of c-Fos, whereas other bursting parameters remain mostly unchanged. By using synthetic TFs with TALE DNA-binding domains, different aspects of these bursts were systematically altered. Specifically, the polymerase initiation frequency was linked to the strength of the transactivation domain and the burst duration to the TF lifetime on the promoter. These results show how TFs and promoter binding domains collectively act to regulate different bursting parameters, offering a vast, evolutionarily tunable regulatory range for individual genes.
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