What's hot today
Saturday, May 31st, 2014
Soza-Ried, C., Ozturk, E., Ish-Horowicz, D. and Lewis, J. (2014). Pulses of Notch activation synchronise oscillating somite cells and entrain the zebrafish segmentation clock. Development 141: 1780-1788. PubMed ID: 24715465
Formation of somites, the rudiments of vertebrate body segments, is an oscillatory process governed by a gene-expression oscillator, the segmentation clock. This operates in each cell of the presomitic mesoderm (PSM), but the individual cells drift out of synchrony when Delta/Notch signalling fails, causing gross anatomical defects. It has been suggested that this is because synchrony is maintained by pulses of Notch activation, delivered cyclically by each cell to its neighbours, that serve to adjust or reset the phase of the intracellular oscillator. This, however, has never been proved. This study provides direct experimental evidence, using zebrafish containing a heat-shock-driven transgene that facilitates delivery of artificial pulses of expression of the Notch ligand DeltaC (see Drosophila Delta). In DeltaC-defective embryos, in which endogenous Notch signalling fails, the artificial pulses restore synchrony, thereby rescuing somite formation. The spacing of segment boundaries produced by repetitive heat-shocking varies according to the time interval between one heat-shock and the next. The induced synchrony is manifest both morphologically and at the level of the oscillations of her1, a core component of the intracellular oscillator. Thus, entrainment of intracellular clocks by periodic activation of the Notch pathway is indeed the mechanism maintaining cell synchrony during somitogenesis.
Saunders, L. R. and McClay, D. R. (2014). Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition. Development 141: 1503-1513. PubMed ID: 24598159
Epithelial-mesenchymal transition (EMT) is a fundamental cell state change that transforms epithelial to mesenchymal cells during embryonic development, adult tissue repair and cancer metastasis. EMT includes a complex series of intermediate cell state changes including remodeling of the basement membrane, apical constriction, epithelial de-adhesion, directed motility, loss of apical-basal polarity, and acquisition of mesenchymal adhesion and polarity. Transcriptional regulatory state changes must ultimately coordinate the timing and execution of these cell biological processes. A well-characterized gene regulatory network (GRN) in the sea urchin embryo was used to identify the transcription factors that control five distinct cell changes during EMT. Single transcription factors were perturbed and the consequences followed with in vivo time-lapse imaging or immunostaining assays. The data show that five different sub-circuits of the GRN control five distinct cell biological activities, each part of the complex EMT process. Thirteen transcription factors (TFs) expressed specifically in pre-EMT cells were required for EMT. Three TFs highest in the GRN specified and activated EMT (alx1, ets1, tbr: see Drosophila Aristaless, Pointed and Optomotor blind/Bifid) and the 10 TFs downstream of those (tel, erg, hex, tgif, snail, twist, foxn2/3, dri, foxb, foxo) were also required for EMT. No single TF functioned in all five sub-circuits, indicating that there is no EMT master regulator. Instead, the resulting sub-circuit topologies suggest EMT requires multiple simultaneous regulatory mechanisms: forward cascades, parallel inputs and positive-feedback lock downs. The interconnected and overlapping nature of the sub-circuits provides one explanation for the seamless orchestration by the embryo of cell state changes leading to successful EMT.
Razy-Krajka, F., Lam, K., Wang, W., Stolfi, A., Joly, M., Bonneau, R. and Christiaen, L. (2014). Collier/OLF/EBF-Dependent Transcriptional Dynamics Control Pharyngeal Muscle Specification from Primed Cardiopharyngeal Progenitors. Dev Cell 29: 263-276. PubMed ID: 24794633
In vertebrates, pluripotent pharyngeal mesoderm progenitors produce the cardiac precursors of the second heart field as well as the branchiomeric head muscles and associated stem cells. However, the mechanisms underlying the transition from multipotent progenitors to distinct muscle precursors remain obscured by the complexity of vertebrate embryos. Using Ciona intestinalis as a simple chordate model, this study shows that bipotent cardiopharyngeal progenitors are primed to activate both heart and pharyngeal muscle transcriptional programs, which progressively become restricted to corresponding precursors. The transcription factor COE (Collier/OLF/EBF; see Drosophila Collier/Knot) orchestrates the transition to pharyngeal muscle fate both by promoting an MRF-associated myogenic program in myoblasts and by maintaining an undifferentiated state in their sister cells through Notch-mediated lateral inhibition. The latter are stem cell-like muscle precursors that form most of the juvenile pharyngeal muscles. The implications of these findings for the development and evolution of the chordate cardiopharyngeal mesoderm are discussed.
Bothe, I., Deng, S. and Baylies, M. (2014). PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site. Development 141(11): 2289-301. PubMed ID: 24821989
Cell-cell fusion is a regulated process that requires merging of the opposing membranes and underlying cytoskeletons. However, the integration between membrane and cytoskeleton signaling during fusion is not known. Using Drosophila, this study demonstrates that the membrane phosphoinositide PI(4,5)P2 is a crucial regulator of F-actin dynamics during myoblast fusion. PI(4,5)P2 is locally enriched and colocalizes spatially and temporally with the F-actin focus that defines the fusion site. PI(4,5)P2 enrichment depends on receptor engagement but is upstream or parallel to actin remodeling. Regulators of actin branching via Arp2/3 (see for example SCAR) colocalize with PI(4,5)P2 in vivo and bind PI(4,5)P2 in vitro. Manipulation of PI(4,5)P2 availability leads to impaired fusion, with a reduction in the F-actin focus size and altered focus morphology. Mechanistically, the changes in the actin focus are due to a failure in the enrichment of actin regulators at the fusion site. Moreover, improper localization of these regulators hinders expansion of the fusion interface. Thus, PI(4,5)P2 enrichment at the fusion site encodes spatial and temporal information that regulates fusion progression through the localization of activators of actin polymerization.
Schrode, N., Saiz, N., Di Talia, S. and Hadjantonakis, A. K. (2014). GATA6 Levels Modulate Primitive Endoderm Cell Fate Choice and Timing in the Mouse Blastocyst. Dev Cell 29: 454-467. PubMed ID: 24835466
Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6 (see Drosophila Serpent), respectively. To investigate the mechanistic regulation of this process, an unbiased, quantitative, single-cell-resolution image analysis pipeline was applied to analyze embryos lacking or exhibiting reduced levels of GATA6. Gata6 mutants were found to exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, it was shown that GATA6 is necessary for PrE specification by FGF signaling, and a model is proposed where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification.
Friday, May 30th
Savoian, M. S. and Glover, D. M. (2014). Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila. Open Biol 4. PubMed ID: 24829288
Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. This study characterizes the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that gamma-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates gamma-tubulin distribution and spindle density. Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together these data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.
Barth, T. K., Schade, G. O., Schmidt, A., Vetter, I., Wirth, M., Heun, P., Thomae, A. W. and Imhof, A. (2014). Identification of novel Drosophila centromere associated proteins. Proteomics [Epub ahead of print]. PubMed ID: 24841622
Centromeres are chromosomal regions crucial for correct chromosome segregation during mitosis and meiosis. They are epigenetically defined by centromeric proteins like the centromere-specific histone H3-variant CENP-A (Centromere identifier or Cid in Drosophila). In humans, 16 additional proteins have been described to be constitutively associated with centromeres throughout the cell cycle, known as the constitutive centromere associated network (CCAN). In contrast, only one additional constitutive centromeric protein is known in Drosophila, the conserved CCAN member CENP-C. To gain further insights into Drosophila centromere composition and biology, affinity-purified chromatin prepared from Drosophila cell lines expressing GFP-tagged H3 variants was analyzed by mass spectrometry. In addition to already known centromeric proteins, novel factors were identified that were repeatedly enriched in affinity purification-MS experiments. The cellular localization of selected candidates was analyzed by immunocytochemistry, and localization to the centromere and other genomic regions was confirmed for ten factors. Furthermore, RNAi-mediated depletion of CG2051 (a histone acetyltransferase type B, catalytic subunit), CG14480 and Hyperplastic discs (Hyd), three of the strongest candidates, leads to elevated mitotic defects. Knockdowns of these candidates neither impair the localization of several known kinetochore proteins nor CENP-A/CID loading, suggesting their involvement in alternative pathways that contribute to proper centromere function. In summary, this study provides a comprehensive analysis of the proteomic composition of Drosophila centromeres.
Iampietro, C., Bergalet, J., Wang, X., Cody, N. A., Chin, A., Lefebvre, F. A., Douziech, M., Krause, H. M. and Lecuyer, E. (2014). Developmentally Regulated Elimination of Damaged Nuclei Involves a Chk2-Dependent Mechanism of mRNA Nuclear Retention. Dev Cell [Epub ahead of print]. PubMed ID: 24835465
The faithful execution of embryogenesis relies on the ability of organisms to respond to genotoxic stress and to eliminate defective cells that could otherwise compromise viability. In syncytial-stage Drosophila embryos, nuclei with excessive DNA damage undergo programmed elimination through an as-yet poorly understood process of nuclear fallout at the midblastula transition. This study shows that this involves a Chk2-dependent mechanism of mRNA nuclear retention that is induced by DNA damage and prevents the translation of specific zygotic mRNAs encoding key mitotic, cytoskeletal, and nuclear proteins required to maintain nuclear viability. For histone messages, this study shows that nuclear retention involves Chk2-mediated inactivation of the Drosophila Stem loop binding protein (SLBP), the levels of which are specifically depleted in damaged nuclei following Chk2 phosphorylation, an event that contributes to nuclear fallout. These results reveal a layer of regulation within the DNA damage surveillance systems that safeguard genome integrity in eukaryotes.
Vazquez-Novelle, M. D., Sansregret, L., Dick, A. E., Smith, C. A., McAinsh, A. D., Gerlich, D. W. and Petronczki, M. (2014). Cdk1 inactivation terminates mitotic checkpoint surveillance and stabilizes kinetochore attachments in anaphase. Curr Biol 24: 638-645. PubMed ID: 24583019
Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome [APC/C(Cdc20)]. Upon satisfaction of both pathways, the APC/C(Cdc20) elicits the degradation of securin (see Drosophila Pimples) and cyclin B. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood. This study shows that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/C(Cdc20) is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. It is proposed that the simultaneous destruction of securin and cyclin B elicited by the APC/C(Cdc20) couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit.
Thursday, May 29th
Yasugi, T., Fischer, A., Jiang, Y., Reichert, H. and Knoblich, J. A. (2014). A regulatory transcriptional loop controls proliferation and differentiation in Drosophila neural stem cells. PLoS One 9: e97034. PubMed ID: 24804774
Neurogenesis is initiated by a set of basic Helix-Loop-Helix (bHLH) transcription factors that specify neural progenitors and allow them to generate neurons in multiple rounds of asymmetric cell division. The Drosophila Daughterless (Da) protein and its mammalian counterparts (E12/E47) act as heterodimerization factors for proneural genes and are therefore critically required for neurogenesis. This study demonstrates that Da can also be an inhibitor of the neural progenitor fate whose absence leads to stem cell overproliferation and tumor formation. This paradox is explained by demonstrating that Da induces the differentiation factor Prospero (Pros) whose asymmetric segregation is essential for differentiation in one of the two daughter cells. Da co-operates with the bHLH transcription factor Asense, whereas the other proneural genes are dispensible. After mitosis, Pros terminates Asense expression in one of the two daughter cells. In da mutants, pros is not expressed, leading to the formation of lethal transplantable brain tumors. The results define a transcriptional feedback loop that regulates the balance between self-renewal and differentiation in Drosophila optic lobe neuroblasts. They indicate that initiation of a neural differentiation program in stem cells is essential to prevent tumorigenesis.
Southall, T. D., Davidson, C. M., Miller, C., Carr, A. and Brand, A. H. (2014). Dedifferentiation of neurons precedes tumor formation in Lola mutants. Dev Cell 28: 685-696. PubMed ID: 24631403
The ability to reprogram differentiated cells into a pluripotent state has revealed that the differentiated state is plastic and reversible. It is evident, therefore, that mechanisms must be in place to maintain cells in a differentiated state. Transcription factors that specify neuronal characteristics have been well studied, but less is known about the mechanisms that prevent neurons from dedifferentiating to a multipotent, stem cell-like state. This study identified Lola as a transcription factor that is required to maintain neurons in a differentiated state. Lola represses neural stem cell genes and cell-cycle genes in postmitotic neurons. In lola mutants, neurons dedifferentiate, turn on neural stem cell genes, and begin to divide, forming tumors. Thus, neurons rather than stem cells or intermediate progenitors are the tumor-initiating cells in lola mutants.
Xiao, N., Privman, E. and Venton, B. J. (2014). Optogenetic control of serotonin and dopamine release in Drosophila larvae. ACS Chem Neurosci [Epub ahead of print]. PubMed ID: 24849718
Optogenetic control of neurotransmitter release is an elegant method to investigate neurobiological mechanisms with millisecond precision and cell type-specific resolution. Channelrhopsin-2 (ChR2) can be expressed in specific neurons and blue light used to activate those neurons. Previously in Drosophila, neurotransmitter release and uptake have been studied after continuous optical illumination. This study investigated the effects of pulsed optical stimulation trains on serotonin or dopamine release in larval ventral nerve cords. In larvae with ChR2 expressed in serotonergic neurons, low-frequency stimulations produced a distinct, steady-state response while high-frequency patterns were peak shaped. Evoked serotonin release increased with increasing stimulation frequency and then plateaued. The steady-state response and the frequency dependence disappeared after administering the uptake inhibitor fluoxetine, indicating that uptake plays a significant role in regulating the extracellular serotonin concentration. Pulsed stimulations were also used to evoke dopamine release in flies expressing ChR2 in dopaminergic neurons and similar frequency dependence was observed. Release due to pulsed optical stimulations was modeled to determine the uptake kinetics. Thus, pulsed optical stimulations can be used to mimic neuronal firing patterns and will allow Drosophila to be used as a model system for studying mechanisms underlying neurotransmission.
Zhou, W., Chang, J., Wang, X., Savelieff, M. G., Zhao, Y., Ke, S. and Ye, B. (2014). GM130 Is Required for Compartmental Organization of Dendritic Golgi Outposts. Curr Biol [Epub ahead of print]. PubMed ID: 24835455
Golgi complexes (Golgi) play important roles in the development and function of neurons. Not only are Golgi present in the neuronal soma (somal Golgi), they also exist in the dendrites as Golgi outposts. Previous studies have shown that Golgi outposts serve as local microtubule-organizing centers and secretory stations in dendrites. It is unknown whether the structure and function of Golgi outposts differ from those of somal Golgi. Here we show in Drosophila that, unlike somal Golgi, the biochemically distinct cis, medial, and trans compartments of Golgi are often disconnected in dendrites in vivo. The Golgi structural protein GM130 is responsible for connecting distinct Golgi compartments in soma and dendritic branch points, and the specific distribution of GM130 determines the compartmental organization of dendritic Golgi in dendritic shafts. It was further shown that compartmental organization regulates the role of Golgi in acentrosomal microtubule growth in dendrites and in dendritic branching. This study provides insights into the structure and function of dendritic Golgi outposts as well as the regulation of compartmental organization of Golgi in general.
Wednesday, May 28th
Delgado, R., Munoz, Y., Pena-Cortes, H., Giavalisco, P. and Bacigalupo, J. (2014). Diacylglycerol Activates the Light-Dependent Channel TRP in the Photosensitive Microvilli of Drosophila melanogaster Photoreceptors. J Neurosci 34: 6679-6686. PubMed ID: 24806693
Drosophila light-dependent channels, TRP and TRPL, reside in the light-sensitive microvilli of the photoreceptor's rhabdomere. Phospholipase C mediates TRP/TRPL opening, but the gating process in visual signal transduction remains unknown. Controversial evidence has suggested diacylglycerol (DAG), polyunsaturated fatty acids (PUFAs, a DAG metabolite), phosphatidylinositol bisphosphate (PIP2), and H(+) as possible channel activators. This study tested each of them directly in inside-out TRP-expressing patches excised from the rhabdomere, making use of mutants and pharmacology. When patches were excised in darkness TRP remained closed, while when excised under illumination it stayed constitutively active. TRP was opened by DAG and silenced by ATP, suggesting DAG-kinase (DGK) involvement. The ATP effect was abolished by inhibiting DGK and in the rdgA mutant, lacking functional DGK, implicating DGK. DAG activated TRP even in the presence of a DAG-lipase inhibitor, inconsistent with a requirement of PUFAs in opening TRP. PIP2 had no effect and acidification, pH 6.4, activated TRP irreversibly, unlike the endogenous activator. Complementary liquid-chromatography/mass-spectrometry determinations of DAG and PUFAs in membranes enriched in rhabdomere obtained from light- and dark-adapted eyes showed light-dependent increment in six DAG species and no changes in PUFAs. The results strongly support DAG as the endogenous TRP agonist, as some of its vertebrate TRPC homologs of the same channel family.
Parsons, L. M., Grzeschik, N. A. and Richardson, H. E. (2014). lgl Regulates the Hippo Pathway Independently of Fat/Dachs, Kibra/Expanded/Merlin and dRASSF/dSTRIPAK. Cancers (Basel) 6: 879-896. PubMed ID: 24743776
In both Drosophila and mammalian systems, the Hippo (Hpo) signalling pathway controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core pathway consists of a protein kinase Hpo (MST1/2 in mammals) that is regulated by a number of upstream inputs including Drosophila Ras Association Factor, dRASSF. Previous studies have shown in the developing Drosophila eye epithelium that loss of the apico-basal cell polarity regulator lethal-(2)-giant-larvae (lgl), and the concomitant increase in aPKC activity, results in ectopic proliferation and suppression of developmental cell death by blocking Hpo pathway signalling. This study further explored how Lgl/aPKC interacts with the Hpo pathway. Deregulation of the Hpo pathway by Lgl depletion is associated with the mislocalization of Hpo and dRASSF. Lgl/aPKC were shown to regulate the Hpo pathway independently of upstream inputs from Fat/Dachs and the Kibra/Expanded/Merlin complex. Depletion of Lgl also results in accumulation and mislocalization of components of the dSTRIPAK complex, a major phosphatase complex that directly binds to dRASSF and represses Hpo activity. However, depleting dSTRIPAK components, or removal of dRASSF did not rescue the lgl-/- or aPKC overexpression phenotypes. Thus, Lgl/aPKC regulate Hpo activity by a novel mechanism, independently of dRASSF and dSTRIPAK. Surprisingly, removal of dRASSF in tissue with increased aPKC activity results in mild tissue overgrowth, indicating that in this context dRASSF acts as a tumor suppressor. This effect was independent of the Hpo and Ras Mitogen Activated Protein Kinase (MAPK) pathways, suggesting that dRASSF regulates a novel pathway to control tissue growth.
Wang, G., Tang, X., Chen, Y., Cao, J., Huang, Q., Ling, X., Ren, W., Liu, S., Wu, Y., Ray, L. and Lin, X. (2014). Hyperplastic Discs Differentially Regulates the Transcriptional Outputs of Hedgehog Signaling. Mech Dev [Epub ahead of print]. PubMed ID: 24854243
Hedgehog (Hh) acts as a morphogen to activate the transcription of diverse target genes via its downstream effector Cubitus interruptus (Ci). Currently, it is less understood how Ci recruits co-factors to activate transcription. This study reports that hyperplastic discs (hyd), an E3 ubiquitin ligase, can differentially regulate the transcriptional outputs of Hh signaling. Loss of Hyd activity caused upregulation of some, but not all of Hh target genes. Importantly, Hyd does not affect the stability of Ci. These data suggest that Hyd differentially restrains the transcriptional activity of Ci via selective association with respective promoters
Reddy, B. A., van der Knaap, J. A., Bot, A. G., Mohd-Sarip, A., Dekkers, D. H., Timmermans, M. A., Martens, J. W., Demmers, J. A. and Verrijzer, C. P. (2014). Nucleotide biosynthetic enzyme GMP synthase is a TRIM21-controlled relay of p53 stabilization. Mol Cell 53: 458-470. PubMed ID: 24462112
Nucleotide biosynthesis is fundamental to normal cell proliferation as well as to oncogenesis. Tumor suppressor p53 (see Drosophila p53), which prevents aberrant cell proliferation, is destabilized through ubiquitylation by MDM2. Ubiquitin-specific protease 7 (USP7) plays a dualistic role in p53 regulation and has been proposed to deubiquitylate either p53 or MDM2. This study shows that guanosine 5'-monophosphate synthase (GMPS) is required for USP7-mediated stabilization of p53. Normally, most GMPS is sequestered in the cytoplasm, separated from nuclear USP7 and p53. In response to genotoxic stress or nucleotide deprivation, GMPS becomes nuclear and facilitates p53 stabilization by promoting its transfer from MDM2 to a GMPS-USP7 deubiquitylation complex. Intriguingly, cytoplasmic sequestration of GMPS requires ubiquitylation by TRIM21, a ubiquitin ligase associated with autoimmune disease. These results implicate a classic nucleotide biosynthetic enzyme and a ubiquitin ligase, better known for its role in autoimmune disease, in p53 control.
Tuesday, May 27th
Driver, I. and Ohlstein, B. (2014). Specification of regional intestinal stem cell identity during Drosophila metamorphosis. Development 141(9): 1848-56. PubMed ID: 24700821
In the adult Drosophila midgut the bone morphogenetic protein (BMP) signaling pathway is required to specify and maintain the acid-secreting region of the midgut known as the copper cell region (CCR). BMP signaling is also involved in the modulation of intestinal stem cell (ISC) proliferation in response to injury. How ISCs are able to respond to the same signaling pathway in a regionally different manner is currently unknown. This study shows that dual use of the BMP signaling pathway in the midgut is possible because BMP signals are capable of transforming ISC and enterocyte identity only during a defined window of metamorphosis. ISC heterogeneity is established prior to adulthood and then maintained in cooperation with regional signals from surrounding tissue. Theses data provide a conceptual framework for how other tissues maintained by regional stem cells might be patterned and establishes the pupal and adult midgut as a novel genetic platform for identifying genes necessary for regional stem cell specification and maintenance.
Chakkalakal, J. V., Christensen, J., Xiang, W., Tierney, M. T., Boscolo, F. S., Sacco, A. and Brack, A. S. (2014). Early forming label-retaining muscle stem cells require p27kip1 for maintenance of the primitive state. Development 141: 1649-1659. PubMed ID: 24715455
Across different niches, subsets of highly functional stem cells are maintained in a relatively dormant rather than proliferative state. Understanding of proliferative dynamics in tissue-specific stem cells during conditions of increased tissue turnover remains limited. Using a TetO-H2B-GFP reporter of proliferative history, skeletal muscle stem cell, or satellite cells, were identified that retain (LRC) or lose (nonLRC) the H2B-GFP label. In mice label-retaining muscle stem cells (LRCs) and nonLRCs are formed at birth and persist during postnatal growth and adult muscle repair. Functionally, LRCs and nonLRCs are born equivalent and transition during postnatal maturation into distinct and hierarchically organized subsets. Adult LRCs give rise to LRCs and nonLRCs; the former are able to self-renew, whereas the latter are restricted to differentiation. Expression analysis revealed the CIP/KIP family members p21(cip1) (Cdkn1a) and p27(kip1) (Cdkn1b) (see Drosophila Dacapo) to be expressed at higher levels in LRCs. In accordance with a crucial role in LRC fate, loss of p27(kip1) promoted proliferation and differentiation of LRCs in vitro and impaired satellite cell self-renewal after muscle injury. By contrast, loss of p21(cip1) only affected nonLRCs, in which myogenic commitment was inhibited. The results provide evidence that restriction of self-renewal potential to LRCs is established early in life and is maintained during increased tissue turnover through the cell cycle inhibitor p27(kip1). They also reveal the differential role of CIP/KIP family members at discrete steps within the stem cell hierarchy.
Konstantinides, N. and Averof, M. (2014). A common cellular basis for muscle regeneration in arthropods and vertebrates. Science 343: 788-791. PubMed ID: 24385602
Many animals are able to regenerate amputated or damaged body parts, but it is unclear whether different taxa rely on similar strategies. Planarians and vertebrates use different strategies, based on pluripotent versus committed progenitor cells, respectively, to replace missing tissues. In most animals, however, experimental tools needed to determine the origin of regenerated tissues are lacking. This study presents a genetically tractable model for limb regeneration, the crustacean Parhyale hawaiensis. Regeneration in Parhyale involves lineage-committed progenitors, as in vertebrates. Pax3/7-expressing muscle satellite cells (See Drosophila Paired), previously identified only in chordates, are shown to be a source of regenerating muscle in Parhyale. These similarities point to a common cellular basis of regeneration, dating back to the common ancestors of bilaterians.
Livigni, A., Peradziryi, H., Sharov, A. A., Chia, G., Hammachi, F., Migueles, R. P., Sukparangsi, W., Pernagallo, S., Bradley, M., Nichols, J., Ko, M. S. and Brickman, J. M. (2013). A Conserved Oct4/POUV-Dependent Network Links Adhesion and Migration to Progenitor Maintenance. Curr Biol 23: 2233-2244. PubMed ID: 24210613
The class V POU domain transcription factor Oct4 (Pou5f1) is a pivotal regulator of embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. Oct4 is also an important evolutionarily conserved regulator of progenitor cell differentiation during embryonic development. This study examined the function of Oct4 homologs in Xenopus embryos and compared this to the role of Oct4 in maintaining mammalian embryo-derived stem cells. Based on a combination of expression profiling of Oct4/POUV-depleted Xenopus embryos and in silico analysis of existing mammalian Oct4 target data sets, a set of evolutionary-conserved Oct4/POUV targets was defined. Most of these targets, were regulators of cell adhesion. This is consistent with Oct4/POUV phenotypes observed in the adherens junctions in Xenopus ectoderm, mouse embryonic, and epiblast stem cells. A number of these targets, including E-cadherin (see Drosophila Shotgun), could rescue both Oct4/POUV phenotypes in cellular adhesion and multipotent progenitor cell maintenance, whereas expression of cadherins on their own could only transiently support adhesion and block differentiation in both ESC and Xenopus embryos. Currently, the list of Oct4 transcriptional targets contains thousands of genes. Using evolutionary conservation, a core set of functionally relevant factors were identified that linked the maintenance of adhesion to Oct4/POUV. It was found that the regulation of adhesion by the Oct4/POUV network occurred at both transcriptional and posttranslational levels and was required for pluripotency.
Monday, May 26th
Choi, B. J., Imlach, W. L., Jiao, W., Wolfram, V., Wu, Y., Grbic, M., Cela, C., Baines, R. A., Nitabach, M. N. and McCabe, B. D. (2014). Miniature neurotransmission regulates Drosophila synaptic structural maturation. Neuron 82: 618-634. PubMed ID: 24811381
Miniature neurotransmission is the transsynaptic process where single synaptic vesicles spontaneously released from presynaptic neurons induce miniature postsynaptic potentials. Since their discovery over 60 years ago, miniature events have been found at every chemical synapse studied. However, the in vivo necessity for these small-amplitude events has remained enigmatic. This study shows that miniature neurotransmission is required for the normal structural maturation of Drosophila glutamatergic synapses in a developmental role that is not shared by evoked neurotransmission. Conversely, it was found that increasing miniature events is sufficient to induce synaptic terminal growth. Miniature neurotransmission acts locally at terminals to regulate synapse maturation via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. These results establish that miniature neurotransmission, a universal but often-overlooked feature of synapses, has unique and essential functions in vivo.
Lepicard, S., Franco, B., de Bock, F. and Parmentier, M. L. (2014). A presynaptic role of microtubule-associated protein 1/futsch in Drosophila: regulation of active zone number and neurotransmitter release. J Neurosci 34: 6759-6771. PubMed ID: 24828631
Structural microtubule-associated proteins (MAPs), like MAP1, not only control the stability of microtubules, but also interact with postsynaptic proteins in the nervous system. Their presynaptic role has barely been studied. To tackle this question, the Drosophila model was used in which there is only one MAP1 homolog, Futsch, which is expressed at the larval neuromuscular junction, presynaptically only. Futsch was shown to regulates neurotransmitter release and active zone density. Importantly, evidence is provided that this role of Futsch is not just the consequence of its microtubule-stabilizing function. Using high-resolution microscopy, Futsch and microtubules were shown to be almost systematically present in close proximity to active zones, with Futsch being localized in-between microtubules and active zones. Using proximity ligation assays, i the proximity of Futsch, but not microtubules, to active zone components was demonstrated. Altogether these data are in favor of a model by which Futsch locally stabilizes active zones, by reinforcing their link with the underlying microtubule cytoskeleton.
Wang, D., Epstein, D., Khalaf, O., Srinivasan, S., Williamson, W. R., Fayyazuddin, A., Quiocho, F. A. and Hiesinger, P. R. (2014). Ca2+-Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1. J Cell Biol 205: 21-31. PubMed ID: 24733584
Most chemical neurotransmission occurs through Ca(2+)-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca(2+) sensing is thought to occur shortly before exocytosis. This paper provides evidence that the Ca(2+) dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca(2+) for the assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) complexes. It was shown that the neuronal -type H(+)-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca(2+)-Calmodulin (CaM)-dependent manner. Ca(2+)-CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca(2+)-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. These data suggest that Ca(2+)-CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.
Devergne, O., Tsung, K., Barcelo, G. and Schupbach, T. (2014). Polarized deposition of basement membrane proteins depends on Phosphatidylinositol synthase and the levels of Phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci U S A. PubMed ID: 24828534
The basement membrane (BM), a specialized sheet of the extracellular matrix contacting the basal side of epithelial tissues, plays an important role in the control of the polarized structure of epithelial cells. However, little is known about how BM proteins themselves achieve a polarized distribution. This study identified phosphatidylinositol 4,5-bisphosphate (PIP2) as a critical regulator of the polarized secretion of BM proteins. A decrease of PIP2 levels, in particular through mutations in Phosphatidylinositol synthase (Pis) and other members of the phosphoinositide pathway, leads to the aberrant accumulation of BM components at the apical side of the cell without primarily affecting the distribution of apical and basolateral polarity proteins. In addition, PIP2 controls the apical and lateral localization of Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein), a factor specifically required to prevent aberrant apical secretion of BM. It is proposed that PIP2, through the control of Crag's subcellular localization, restricts the secretion of BM proteins to the basal side.
Sunday, May 25th
Serralbo, O. and Marcelle, C. (2014). Migrating cells mediate long-range WNT signaling. Development 141: 2057-2063. PubMed ID: 24803654
In amniotes, it is widely accepted that WNTs secreted by the dorsal neural tube form a concentration gradient that regulates early somite patterning and myotome organization. This study demonstrates in the chicken embryo that WNT protein (See Drosophila Wingless) is not secreted to act at a distance, but rather loaded onto migrating neural crest cells that deliver it to somites. Inhibiting neural crest migration or ablating their population has a profound impact on the WNT response in somites. Furthermore, it was shown that a central player in the efficient delivery of WNT to somites is the heparan sulfate proteoglycan GPC4, expressed by neural crest. Together, these data describe a novel mode of signaling whereby WNT proteins hitch a ride on migratory neural crest cells to pattern the somites at a distance from its source.
Hung, W. L., Wang, Y., Chitturi, J. and Zhen, M. (2014). A Caenorhabditis elegans developmental decision requires insulin signaling-mediated neuron-intestine communication. Development 141: 1767-1779. PubMed ID: 24671950
Adverse environmental conditions trigger C. elegans larvae to activate an alternative developmental program, termed dauer diapause, which renders them stress resistant. High-level insulin signaling prevents constitutive dauer formation. However, it is not fully understood how animals assess conditions to choose the optimal developmental program. This study shows that insulin-like peptide (ILP)-mediated neuron-intestine communication plays a role in this developmental decision. Consistent with, and extending, previous findings, it was shown that the simultaneous removal of INS-4, INS-6 and DAF-28 leads to fully penetrant constitutive dauer formation, whereas the removal of INS-1 and INS-18 significantly inhibits constitutive dauer formation. These ligands are processed by the proprotein convertases PC1/KPC-1 and/or PC2/EGL-3. The agonistic and antagonistic ligands are expressed by, and function in, neurons to prevent or promote dauer formation. By contrast, the insulin receptor DAF-2 (see Drosophila Insulin-like receptor) and its effector, the FOXO transcription factor DAF-16 (see Drosophila Foxo), function solely in the intestine to regulate the decision to enter diapause. These results suggest that the nervous system normally establishes an agonistic ILP-dominant paradigm to inhibit intestinal DAF-16 activation and allow reproductive development. Under adverse conditions, a switch in the agonistic-antagonistic ILP balance activates intestinal DAF-16, which commits animals to diapause.
Zhang, W., Cheng, Y., Li, Y., Chen, Z., Jin, P. and Chen, D. (2014). A feed-forward mechanism involving Drosophila fragile X mental retardation protein triggers a replication stress-induced DNA damage response. Hum Mol Genet [Epub ahead of print]. PubMed ID: 24833720
Fragile X syndrome, a common form of inherited mental retardation, is caused by loss of the fragile X mental retardation protein (FMRP). As a selective RNA-binding protein, FMRP is localized predominately in cytoplasm, where it regulates translational control. However, there is a small portion of FMRP present in the nucleus, and its function there has been elusive. This study shows that Drosophila dFMR1 in nucleus is required for replication stress-induced H2Av phosphorylation in the DNA damage response (DDR). Replication stress could induce the expression of dFmr1 and promote the nuclear accumulation of dFMR1. Upon the stimulation of replication stress, dFMR1 is associated with chromatin in a domain-specific manner, which is essential for its ability to induce the phosphorylation of H2Av. These results together reveal an unexpected nuclear role of FMRP in DDR and uncover a feed-forward mechanism by which dFmr1 and early DDR induced by replication stress reciprocally regulate each other, thereby synergistically triggering activity of the DDR signaling cascade.
Waterson, M. J., Chung, B. Y., Harvanek, Z. M., Ostojic, I., Alcedo, J. and Pletcher, S. D. (2014). Water sensor ppk28 modulates Drosophila lifespan and physiology through AKH signaling. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 24821805
Sensory perception modulates lifespan across taxa, presumably due to alterations in physiological homeostasis after central nervous system integration. The coordinating circuitry of this control, however, remains unknown. This study used the Drosophila melanogaster gustatory system to dissect one component of sensory regulation of aging. Loss of the critical water sensor, pickpocket 28 (ppk28), altered metabolic homeostasis to promote internal lipid and water stores and extended healthy lifespan. Additionally, loss of ppk28 increased neuronal glucagon-like adipokinetic hormone (AKH) signaling, and the AKH receptor was necessary for ppk28 mutant effects. Furthermore, activation of AKH-producing cells alone was sufficient to enhance longevity, suggesting that a perceived lack of water availability triggers a metabolic shift that promotes the production of metabolic water and increases lifespan via AKH signaling. This work provides an example of how discrete gustatory signals recruit nutrient-dependent endocrine systems to coordinate metabolic homeostasis, thereby influencing long-term health and aging.
Saturday, May 24th
Parrish, J. Z., Kim, C. C., Tang, L., Bergquist, S., Wang, T., Derisi, J. L., Jan, L. Y., Jan, Y. N. and Davis, G. W. (2014). Kruppel mediates the selective rebalancing of ion channel expression. Neuron 82: 537-544. PubMed ID: 24811378
Ion channel gene expression can vary substantially among neurons of a given type, even though neuron-type-specific firing properties remain stable and reproducible. The mechanisms that modulate ion channel gene expression and stabilize neural firing properties are unknown. In Drosophila, this study demonstrates that loss of the Shal potassium channel induces the compensatory rebalancing of ion channel expression including, but not limited to, the enhanced expression and function of Shaker and slowpoke. Using genomic and network modeling approaches combined with genetic and electrophysiological assays, this study demonstrates that the transcription factor Kruppel is necessary for the homeostatic modulation of Shaker and slowpoke expression. Remarkably, Kruppel induction is specific to the loss of Shal, not being observed in five other potassium channel mutants that cause enhanced neuronal excitability. Thus, homeostatic signaling systems responsible for rebalancing ion channel expression can be selectively induced after the loss or impairment of a specific ion channel.
>Rowshanravan, B., Woodcock, S. A., Botella, J. A., Kiermayer, C., Schneuwly, S. and Hughes, D. A. (2014). RasGAP mediates neuronal survival in Drosophila through direct regulation of Rab5-dependent endocytosis. J Cell Sci. PubMed ID: 24816559
The GTPase Ras can either promote or inhibit cell survival. Inactivating mutations in RasGAP [vacuolar peduncle (vap)], a Ras GTPase-activating protein, lead to age-related brain degeneration in Drosophila. Genetic interactions implicate the Epidermal growth factor receptor (EGFR)-Ras pathway in promoting neurodegeneration but the mechanism is not known. This study shows that the Src homology 2 (SH2) domains of RasGAP are essential for its neuroprotective function. By using affinity purification and mass spectrometry, a complex was identified containing RasGAP together with Sprint, a Ras effector and putative activator of the endocytic GTPase Rab5. Formation of the RasGAP-Sprint complex requires the SH2 domains of RasGAP and tyrosine phosphorylation of Sprint. RasGAP and Sprint co-localize with Rab5-positive early endosomes but not with Rab7-positive late endosomes. This study demonstrates a key role for this interaction in neurodegeneration: mutation of Sprint (or Rab5) suppresses neuronal cell death caused by the loss of RasGAP. These results indicate that the long-term survival of adult neurons in Drosophila is critically dependent on the activities of two GTPases, Ras and Rab5, regulated by the interplay of RasGAP and Sprint.
Omi, M., Harada, H., Watanabe, Y., Funahashi, J. and Nakamura, H. (2014). Role of En2 in the tectal laminar formation of chick embryos. Development 141: 2131-2138. PubMed ID: 24803658
The chick optic tectum consists of 16 laminae. This study reports contribution of En2 to laminar formation in chick optic tecta. En2 (see Drosophila Engrailed) is specifically expressed in laminae g-j of stratum griseum et fibrosum superficiale (SGFS). Misexpression of En2 resulted in disappearance of En2-expressing cells from the superficial layers (laminae a-f of SGFS), where endogenous En2 is not expressed. Misexpression of En2 before postmitotic cells had left the ventricular layer indicated that En2-misexpressing cells stopped at the laminae of endogenous En2 expression and that they did not migrate into the superficial layers. Induction of En2 misexpression using a tetracycline-inducible system after the postmitotic cells had reached superficial layers also resulted in disappearance of En2-expressing cells from the superficial layers. Time-lapse analysis showed that En2-misexpressing cells migrated back from the superficial layers towards the middle layers, where En2 is strongly expressed endogenously. These results suggest a potential role of En2 in regulating cell migration and positioning in the tectal laminar formation.
Kunte, K., Zhang, W., Tenger-Trolander, A., Palmer, D. H., Martin, A., Reed, R. D., Mullen, S. P. and Kronforst, M. R. (2014). doublesex is a mimicry supergene. Nature 507: 229-232. PubMed ID: 24598547
One of the most striking examples of sexual dimorphism is sex-limited mimicry in butterflies, a phenomenon in which one sex--usually the female--mimics a toxic model species, whereas the other sex displays a different wing pattern. Sex-limited mimicry is phylogenetically widespread in the swallowtail butterfly genus Papilio, in which it is often associated with female mimetic polymorphism. In multiple polymorphic species, the entire wing pattern phenotype is controlled by a single Mendelian 'supergene'. Although theoretical work has explored the evolutionary dynamics of supergene mimicry, there are almost no empirical data that address the critical issue of what a mimicry supergene actually is at a functional level. Using an integrative approach combining genetic and association mapping, transcriptome and genome sequencing, and gene expression analyses, this study shows that a single gene, doublesex (homolog of Drosophila doublesex), controls supergene mimicry in Papilio polytes. This is in contrast to the long-held view that supergenes are likely to be controlled by a tightly linked cluster of loci. Analysis of gene expression and DNA sequence variation indicates that isoform expression differences contribute to the functional differences between dsx mimicry alleles, and protein sequence evolution may also have a role. The results combine elements from different hypotheses for the identity of supergenes, showing that a single gene can switch the entire wing pattern among mimicry phenotypes but may require multiple, tightly linked mutations to do so.
Friday, May 23rd
Amandio, A. R., Gaspar, P., Whited, J. L. and Janody, F. (2014). Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels. PLoS One 9: e96326. PubMed ID: 24788460
The actin-Capping Protein heterodimer, composed of the alpha and beta subunits (Capping protein alpha and Capping protein beta), is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. This study aimed to understand how both subunits regulate each other in vivo. The levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein alpha and beta decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. It is proposed that the ability of the alpha and beta subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth.
Hain, D., Langlands, A., Sonnenberg, H. C., Bailey, C., Bullock, S. L. and Muller, H. A. (2014). The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport. Development 141: 2119-2130. PubMed ID: 24803657
Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. This study shows that mutations in drop out (dop), CG6498, disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein.
Salvany, L., Muller, J., Guccione, E. and Rorth, P. (2014). The core and conserved role of MAL is homeostatic regulation of actin levels. Genes Dev 28: 1048-1053. PubMed ID: 24831700
The transcription cofactor MAL (Myocardin-related transcription factor or Mrtf) is regulated by free actin levels and thus by actin dynamics. MAL, together with its DNA-binding partner, SRF, is required for invasive cell migration and in experimental metastasis. Although MAL/SRF has many targets, this study provides genetic evidence in both Drosophila and human cellular models that actin is the key target that must be regulated by MAL/SRF for invasive cell migration. By regulating MAL/SRF activity, actin protein feeds back on production of actin mRNA to ensure sufficient supply of actin. This constitutes a dedicated homeostatic feedback system that provides a foundation for cellular actin dynamics.
Fischer, S. C., Blanchard, G. B., Duque, J., Adams, R. J., Arias, A. M., Guest, S. D. and Gorfinkiel, N. (2014). Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics. PLoS One 9: e95695. PubMed ID: 24759936
Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. This study combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. A decrease in Myosin phosphorylation was found to induce a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. It is suggested that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through E-Cadherin-mediated adhesion. Altogether, these results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.
Thursday, May 22nd
Ha, T. S., Xia, R., Zhang, H., Jin, X. and Smith, D. P. (2014). Lipid flippase modulates olfactory receptor expression and odorant sensitivity in Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 24821794
In Drosophila melanogaster, the male-specific pheromone cVA (11-cis-vaccenyl acetate) functions as a sex-specific social cue. However, understanding of the molecular mechanisms underlying cVA pheromone transduction and its regulation are incomplete. Using a genetic screen combined with an electrophysiological assay to monitor pheromone-evoked activity in the cVA-sensing Or67d neurons, an olfactory sensitivity factor was identified encoded by the dATP8B gene, the Drosophila homolog of mammalian ATP8B. dATP8B is expressed in all olfactory neurons that express Orco, the odorant receptor coreceptor, and the odorant responses in most Orco-expressing neurons are reduced. Or67d neurons are severely affected, with strongly impaired cVA-induced responses and lacking spontaneous spiking in the mutants. The dATP8B locus encodes a member of the P4-type ATPase family thought to flip aminophospholipids such as phosphatidylserine and phosphatidylethanolamine from one membrane leaflet to the other. dATP8B protein is concentrated in the cilia of olfactory neuron dendrites, the site of odorant transduction. Focusing on Or67d neuron function, this study showed that Or67d receptors are mislocalized in dATP8B mutants and that cVA responses can be restored to dATP8B mutants by misexpressing a wild-type dATP8B rescuing transgene, by expressing a vertebrate P4-type ATPase member in the pheromone-sensing neurons or by overexpressing Or67d receptor subunits. These findings reveal an unexpected role for lipid translocation in olfactory receptor expression and sensitivity to volatile odorants.
Grygoruk, A., Chen, A., Martin, C. A., Lawal, H. O., Fei, H., Gutierrez, G., Biedermann, T., Najibi, R., Hadi, R., Chouhan, A. K., Murphy, N. P., Schweizer, F. E., Macleod, G. T., Maidment, N. T. and Krantz, D. E. (2014). The redistribution of Drosophila vesicular monoamine transporter mutants from synaptic vesicles to large dense-core vesicles impairs amine-dependent behaviors. J Neurosci 34: 6924-6937. PubMed ID: 24828646
Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, this study generated mutations in the C-terminal trafficking domain of the Drosophila vesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs. Deletion of the terminal 23 aa (DVMAT-Delta3) reduced the rate of endocytosis and localization of DVMAT to SVs, but supported localization to LDCVs. An alanine substitution mutation in a tyrosine-based motif (DVMAT-Y600A) also reduced sorting to SVs and showed an endocytic deficit specific to aminergic nerve terminals. Redistribution of DVMAT-Y600A from SV to LDCV fractions was also enhanced in aminergic neurons. To determine how these changes might affect behavior, DVMAT-Delta3 and DVMAT-Y600A were expressed in a dVMAT null genetic background that lacks endogenous dVMAT activity. When expressed ubiquitously, DVMAT-Delta3 showed a specific deficit in female fertility, whereas DVMAT-Y600A rescued behavior similarly to DVMAT-wt. In contrast, when expressed more specifically in octopaminergic neurons, both DVMAT-Delta3 and DVMAT-Y600A failed to rescue female fertility, and DVMAT-Y600A showed deficits in larval locomotion. DVMAT-Y600A also showed more severe dominant effects than either DVMAT-wt or DVMAT-Delta3. It is proposed that these behavioral deficits result from the redistribution of DVMAT from SVs to LDCVs. By extension, the data suggest that the balance of amine release from SVs versus that from LDCVs is critical for the function of some aminergic circuits.
Velez, M. M., Wernet, M. F., Clark, D. A. and Clandinin, T. R. (2014). Walking Drosophila align with the e-vector of linearly polarized light through directed modulation of angular acceleration. J Comp Physiol A Neuroethol Sens Neural Behav Physiol [Epub ahead of print]. PubMed ID: 24810784
Understanding the mechanisms that link sensory stimuli to animal behavior is a central challenge in neuroscience. The quantitative description of behavioral responses to defined stimuli has led to a rich understanding of different behavioral strategies in many species. One important navigational cue perceived by many vertebrates and insects is the e-vector orientation of linearly polarized light. Drosophila manifests an innate orientation response to this cue ('polarotaxis'), aligning its body axis with the e-vector field. This study established a population-based behavioral paradigm for the genetic dissection of neural circuits guiding polarotaxis to both celestial as well as reflected polarized stimuli. However, the behavioral mechanisms by which flies align with a linearly polarized stimulus remain unknown. This study presents a detailed quantitative description of Drosophila polarotaxis, systematically measuring behavioral parameters that are modulated by the stimulus. Angular acceleration is shown to be modulated during alignment, and this single parameter may be sufficient for alignment. Furthermore, using monocular deprivation, it was shown that each eye is necessary for modulating turns in the ipsilateral direction. This analysis lays the foundation for understanding how neural circuits guide these important visual behaviors.
Saleem, S., Ruggles, P. H., Abbott, W. K. and Carney, G. E. (2014). Sexual Experience Enhances Drosophila melanogaster Male Mating Behavior and Success. PLoS One 9: e96639. PubMed ID: 24805129
Competition for mates is a wide-spread phenomenon affecting individual reproductive success. The ability of animals to adjust their behaviors in response to changing social environment is important and well documented. Drosophila melanogaster males compete with one another for matings with females and modify their reproductive behaviors based on prior social interactions. However, it remains to be determined how male social experience that culminates in mating with a female impacts subsequent male reproductive behaviors and mating success. This study shows that sexual experience enhances future mating success. Previously mated D. melanogaster males adjust their courtship behaviors and out-compete sexually inexperienced males for copulations. Interestingly, courtship experience alone is not sufficient in providing this competitive advantage, indicating that copulation plays a role in reinforcing this social learning. It was also shown that females use their sense of hearing to preferentially mate with experienced males when given a choice. These results demonstrate the ability of previously mated males to learn from their positive sexual experiences and adjust their behaviors to gain a mating advantage. These experienced-based changes in behavior reveal strategies that animals likely use to increase their fecundity in natural competitive environments.
Wednesday, May 21st
Weber, C. M. Ramachandran, S. and Henikoff, S. (2014). Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol Cell 53: 819-830. PubMed ID: 24606920
Nucleosomes are barriers to transcription in vitro; however, their effects on RNA polymerase in vivo are unknown. This study describes a simple and general strategy to comprehensively map the positions of elongating and arrested RNA polymerase II (RNAPII) at nucleotide resolution. The entry site of the first (+1) nucleosome was found to be a barrier to RNAPII for essentially all genes, including those undergoing regulated pausing farther upstream. In contrast to the +1 nucleosome, gene body nucleosomes are low barriers and cause RNAPII stalling both at the entry site and near the dyad axis. The extent of the +1 nucleosome barrier correlates with nucleosome occupancy but anticorrelates with enrichment of histone variant H2A.Z. Importantly, depletion of H2A.Z from a nucleosome position results in a higher barrier to RNAPII. These results suggest that nucleosomes present significant, context-specific barriers to RNAPII in vivo that can be tuned by the incorporation of H2A.Z.
Thomas, C. J., Kotova, E., Andrake, M., Adolf-Bryfogle, J., Glaser, R., Regnard, C. and Tulin, A. V. (2014). Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation via poly(ADP-ribosyl)ation. Mol Cell 53: 831-842. PubMed ID: 24508391
Dynamically controlled posttranslational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. A nuclear tandem kinase, JIL-1, controls gene expression by activating poly(ADP-ribose) polymerase-1 (PARP-1). JIL-1 phosphorylates the C terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch, consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch, leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. It is proposed that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1-dependent genes.
Dottermusch-Heidel, C., Gartner, S. M., Tegeder, I., Rathke, C., Barckmann, B., Bartkuhn, M., Bhushan, S., Steger, K., Meinhardt, A. and Renkawitz-Pohl, R. (2014). H3K79 methylation: a new conserved mark that accompanies H4 hyperacetylation prior to histone-to-protamine transition in Drosophila and rat. Biol Open. PubMed ID: 24795146
During spermiogenesis, haploid spermatids undergo extensive chromatin remodeling events in which histones are successively replaced by more basic protamines to generate highly compacted chromatin. This study shows that H3K79 methylation is a conserved feature preceding the histone-to-protamine transition in Drosophila melanogaster and rat. During Drosophila spermatogenesis, the Dot1-like methyltransferase Grappa (Gpp) is primarily expressed in canoe stage nuclei. The corresponding H3K79 methylation is a histone modification that precedes the histone-to-protamine transition and correlates with histone H4 hyperacetylation. When acetylation was inhibited in cultured Drosophila testes, nuclei were smaller and chromatin was compact, Gpp was little synthesized, H3K79 methylation was strongly reduced, and protamines were not synthesized. The Gpp isoform Gpp-D has a unique C-terminus, and Gpp is essential for full fertility. In rat, H3K79 methylation also correlates with H4 hyperacetylation but not with active RNA polymerase II, which might point towards a conserved function in chromatin remodeling during the histone-to-protamine transition in both Drosophila and rat.
Marr, S. K., Lis, J. T., Treisman, J. E. and Marr, M. T. (2014). The Metazoan-Specific Mediator Subunit 26 (Med26) Is Essential For Viability And Is Found At Both Active Genes And Pericentric Heterochromatin In Drosophila. Mol Cell Biol. PubMed ID: 24820420
Human Med26 was originally purified in the Cofactor Required for Sp1 Activation complex (CRSP) as a 70 kilodalton component named CRSP70. This polypeptide was specific to metazoans and the 'small' form of the Mediator complex. This paper report that a Drosophila homologue of Med26 similarly interacts with other components of the core Drosophila Mediator complex but not with the kinase module, and is recruited to genes upon activation. Using a null allele of Med26, it was shown that Med26 is required for organismal viability but not for cell proliferation or survival. Clones lacking Med26 in the wing disc lead to loss of the adult wing margin and reduced expression of genes involved in wing margin formation. Surprisingly, when polytene chromosomes from the salivary gland were examined using antibodies to Med26, it was apparent that a fraction of the protein is associated with the chromocenter, which contains pericentric heterochromatin. This staining co-localizes with heterochromatin protein 1 (HP1). Immunoprecipitation experiments show that Med26 interacts with HP1. The interaction is mediated through the chromoshadow domain of HP1 and through the conserved motif in the carboxy-terminus of the Med26 protein. This work is the first characterization of the metazoan-specific Mediator subunit in an animal model.
Tuesday, May 20th
Rumpf, S., Bagley, J. A., Thompson-Peer, K. L., Zhu, S., Gorczyca, D., Beckstead, R. B., Jan, L. Y. and Jan, Y. N. (2014). Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 24799714
The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process-also known as dendrite pruning-depends on the ubiquitin-proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. This study shows that mutation or inhibition of Valosin-Containing Protein (VCP; termed TER94 by FlyBase), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, it was found that VCP inhibition causes an altered splicing pattern of the large pruning gene Molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). These data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 (Effete) and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, these results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism.
Medioni, C., Ramialison, M., Ephrussi, A. and Besse, F. (2014). Imp Promotes Axonal Remodeling by Regulating profilin mRNA during Brain Development. Curr Biol 24: 793-800. PubMed ID: 24656828
Neuronal remodeling is essential for the refinement of neuronal circuits in response to developmental cues. Although this process involves pruning or retraction of axonal projections followed by axonal regrowth and branching, how these steps are controlled is poorly understood. Drosophila mushroom body (MB) gamma neurons provide a paradigm for the study of neuronal remodeling, as their larval axonal branches are pruned during metamorphosis and re-extend to form adult-specific branches. This study identified the RNA binding protein Imp as a key regulator of axonal remodeling. Imp is the sole fly member of a conserved family of proteins that bind target mRNAs to promote their subcellular targeting. Whereas Imp is dispensable for the initial growth of MB gamma neuron axons, it is required for the regrowth and ramification of axonal branches that have undergone pruning. Furthermore, Imp is actively transported to axons undergoing developmental remodeling. Finally, this study demonstrates that profilin mRNA is a direct and functional target of Imp that localizes to axons and controls axonal regrowth. This study reveals that mRNA localization machineries are actively recruited to axons upon remodeling and suggests a role of mRNA transport in developmentally programmed rewiring of neuronal circuits during brain maturation.
He, H., Kise, Y., Izadifar, A., Urwyler, O., Ayaz, D., Parthasarthy, A., Yan, B., Erfurth, M. L., Dascenco, D. and Schmucker, D. (2014). Cell-Intrinsic Requirement of Dscam1 Isoform Diversity for Axon Collateral Formation. Science [Epub ahead of print]. PubMed ID: 24831526
The isoform diversity of the Drosophila Dscam1 receptor is important for neuronal self-recognition and self-avoidance. A canonical model suggests that homophilic binding of identical Dscam1 receptor isoforms on sister dendrites ensures self-avoidance even when just a single isoform is expressed. This study reports on a cell-intrinsic function requiring the co-expression of multiple isoforms. Manipulation of the Dscam1 isoform pool in single neurons causes severe disruption of collateral formation of mechanosensory axons. Changes in isoform abundance lead to dominant dosage sensitive inhibition of branching. It is proposed that the ratio of matching to non-matching isoforms within a cell influences the Dscam1-mediated signaling strength controlling axon growth and growth-cone sprouting. Cell-intrinsic use of surface receptor diversity may be of general importance in regulating axonal branching during brain wiring.
Rawson, R. L., Yam, L., Weimer, R. M., Bend, E. G., Hartwieg, E., Horvitz, H. R., Clark, S. G. and Jorgensen, E. M. (2014). Axons Degenerate in the Absence of Mitochondria in C. elegans. Curr Biol 24: 760-765. PubMed ID: 24631238
Many neurodegenerative disorders are associated with mitochondrial defects. Mitochondria can play an active role in degeneration by releasing reactive oxygen species and apoptotic factors. Alternatively, mitochondria can protect axons from stress and insults, for example by buffering calcium. Recent studies manipulating mitochondria lend support to both of these models. This study identified a C. elegans mutant, ric-7, in which mitochondria are unable to exit the neuron cell bodies, similar to the kinesin-1/unc-116 mutant. When axons lacking mitochondria are cut with a laser, they rapidly degenerate. Some neurons even spontaneously degenerate in ric-7 mutants. Degeneration can be suppressed by forcing mitochondria into the axons of the mutants. The protective effect of mitochondria is also observed in the wild-type: a majority of axon fragments containing a mitochondrion survive axotomy, whereas those lacking mitochondria degenerate. Thus, mitochondria are not required for axon degeneration and serve a protective role in C. elegans axons.
Monday, May 19th
Hatakeyama, J., Wakamatsu, Y., Nagafuchi, A., Kageyama, R., Shigemoto, R. and Shimamura, K. (2014). Cadherin-based adhesions in the apical endfoot are required for active Notch signaling to control neurogenesis in vertebrates. Development 141: 1671-1682. PubMed ID: 24715457
The development of the vertebrate brain requires an exquisite balance between proliferation and differentiation of neural progenitors. Notch signaling plays a pivotal role in regulating this balance, yet the interaction between signaling and receiving cells remains poorly understood. This study found that numerous nascent neurons and/or intermediate neurogenic progenitors expressing the ligand of Notch retain apical endfeet transiently at the ventricular lumen that form adherens junctions (AJs) with the endfeet of progenitors. Forced detachment of the apical endfeet of those differentiating cells by disrupting AJs resulted in precocious neurogenesis that was preceded by the downregulation of Notch signaling. Both Notch1 and its ligand Dll1 are distributed around AJs in the apical endfeet, and these proteins physically interact with ZO-1 (see Drosophila Polychaetoid), a constituent of the AJ. Furthermore, live imaging of a fluorescently tagged Notch1 demonstrated its trafficking from the apical endfoot to the nucleus upon cleavage. These results identified the apical endfoot as the central site of active Notch signaling to securely prohibit inappropriate differentiation of neural progenitors.
Young, J. J., Kjolby, R. A., Kong, N. R., Monica, S. D. and Harland, R. M. (2014). Spalt-like 4 promotes posterior neural fates via repression of pou5f3 family members in Xenopus. Development 141: 1683-1693. PubMed ID: 24715458
Amphibian neural development occurs as a two-step process: (1) induction specifies a neural fate in undifferentiated ectoderm; and (2) transformation induces posterior spinal cord and hindbrain. Signaling through the Fgf, retinoic acid (RA) and Wnt/beta-catenin pathways is necessary and sufficient to induce posterior fates in the neural plate, yet a mechanistic understanding of the process is lacking. This study screened for factors enriched in posterior neural tissue and identified spalt-like 4 (sall4) (see Drosophila Spalt major), which is induced by Fgf. Knockdown of Sall4 results in loss of spinal cord marker expression and increased expression of pou5f3.2 (oct25), pou5f3.3 (oct60) and pou5f3.1 (oct91) (collectively, pou5f3 genes), the closest Xenopus homologs of mammalian stem cell factor Pou5f1 (Oct4). Overexpression of the pou5f3 genes results in the loss of spinal cord identity and knockdown of pou5f3 function restores spinal cord marker expression in Sall4 morphants. Finally, knockdown of Sall4 blocks the posteriorizing effects of Fgf and RA signaling in the neurectoderm. These results suggest that Sall4, activated by posteriorizing signals, represses the pou5f3 genes to provide a permissive environment allowing for additional Wnt/Fgf/RA signals to posteriorize the neural plate.
Duval, N., Daubas, P., Bourcier de Carbon, C., St Cloment, C., Tinevez, J. Y., Lopes, M., Ribes, V. and Robert, B. (2014). Msx1 and Msx2 act as essential activators of Atoh1 expression in the murine spinal cord. Development 141: 1726-1736. PubMed ID: 24715462
Dorsal spinal neurogenesis is orchestrated by the combined action of signals secreted from the roof plate organizer and a downstream transcriptional cascade. Within this cascade, Msx1 and Msx2 (homologs of Drosophila Drop), two homeodomain transcription factors (TFs), are induced earlier than bHLH neuralizing TFs. Whereas bHLH TFs have been shown to specify neuronal cell fate, the function of Msx genes remains poorly defined. This study describes dramatic alterations of neuronal patterning in Msx1/Msx2 double-mutant mouse embryos. The most dorsal spinal progenitor pool fails to express the bHLH neuralizing TF Atoh1 (see Drosophila Atonal), which results in a lack of Lhx2-positive and Barhl2-positive dI1 interneurons. Neurog1 and Ascl1 expression territories are dorsalized, leading to ectopic dorsal differentiation of dI2 and dI3 interneurons. In proportion, the amount of Neurog1-expressing progenitors appears unaffected, whereas the number of Ascl1-positive cells is increased. These defects occur while BMP signaling is still active in the Msx1/Msx2 mutant embryos. Cell lineage analysis and co-immunolabeling demonstrate that Atoh1-positive cells derive from progenitors expressing both Msx1 and Msx2. In vitro, Msx1 and Msx2 proteins activate Atoh1 transcription by specifically interacting with several homeodomain binding sites in the Atoh1 3' enhancer. In vivo, Msx1 and Msx2 are required for Atoh1 3' enhancer activity and ChIP experiments confirm Msx1 binding to this regulatory sequence. These data support a novel function of Msx1 and Msx2 as transcriptional activators. This study provides new insights into the transcriptional control of spinal cord patterning by BMP signaling, with Msx1 and Msx2 acting upstream of Atoh1.
Prin, F., Serpente, P., Itasaki, N. and Gould, A. P. (2014). Hox proteins drive cell segregation and non-autonomous apical remodelling during hindbrain segmentation. Development 141: 1492-1502. PubMed ID: 24574009
Hox genes encode a conserved family of homeodomain transcription factors regulating development along the major body axis. During embryogenesis, Hox proteins are expressed in segment-specific patterns and control numerous different segment-specific cell fates. It has been unclear, however, whether Hox proteins drive the epithelial cell segregation mechanism that is thought to initiate the segmentation process. This study investigated the role of vertebrate Hox proteins during the partitioning of the developing hindbrain into lineage-restricted units called rhombomeres. Loss-of-function mutants and ectopic expression assays reveal that Hoxb4 and its paralogue Hoxd4 (homologs of Drosophila Deformed) are necessary and sufficient for cell segregation, and for the most caudal rhombomere boundary (r6/r7). Hox4 proteins regulate Eph/ephrins and other cell-surface proteins, and can function in a non-cell-autonomous manner to induce apical cell enlargement on both sides of their expression border. Similarly, other Hox proteins expressed at more rostral rhombomere interfaces can also regulate Eph/ephrins (see Drosophila Ephrin), induce apical remodelling and drive cell segregation in ectopic expression assays. However, Krox20, a key segmentation factor expressed in odd rhombomeres (r3 and r5), can largely override Hox proteins at the level of regulation of a cell surface target, Epha4. This study suggests that most, if not all, Hox proteins share a common potential to induce cell segregation but in some contexts this is masked or modulated by other transcription factors.
Sunday, May 18th
Vaughan, A. G., Zhou, C., Manoli, D. S. and Baker, B. S. (2014). Neural Pathways for the Detection and Discrimination of Conspecific Song in D. melanogaster. Curr Biol [Epub ahead of print]. PubMed ID: 24794294
During courtship, male Drosophila melanogaster sing a multipart courtship song to female flies. This song is of particular interest because (1) it is species specific and varies widely within the genus, (2) it is a gating stimulus for females, who are sensitive detectors of conspecific song, and (3) it is the only sexual signal that is under both neural and genetic control. This song is perceived via mechanosensory neurons in the antennal Johnston's organ, which innervate the antennal mechanosensory and motor center (AMMC) of the brain. However, AMMC outputs that are responsible for detection and discrimination of conspecific courtship song remain unknown. Using a large-scale anatomical screen of AMMC interneurons, this study identified seven projection neurons (aPNs) and five local interneurons (aLNs) that outline a complex architecture for the ascending mechanosensory pathway. Neuronal inactivation and hyperactivation during behavior reveal that only two classes of interneurons are necessary for song responses-the projection neuron aPN1 and GABAergic interneuron aLN(al). These neurons are necessary in both male and female flies. Physiological recordings in aPN1 reveal the integration of courtship song as a function of pulse rate and outline an intracellular transfer function that likely facilitates the response to conspecific song. These results reveal a critical pathway for courtship hearing in male and female flies, in which both aLN(al) and aPN1 mediate the detection of conspecific song. The pathways arising from these neurons likely serve as a critical neural substrate for behavioral reproductive isolation in D. melanogaster.
Williams, M. J., Goergen, P., Phad, G., Fredriksson, R. and Schioth, H. B. (2014). The Drosophila Kctd-family homologue Kctd12-like modulates male aggression and mating behaviour. Eur J Neurosci. PubMed ID: 24830553
In Drosophila, serotonin (5-HT) regulates aggression, mating behaviour and sleep/wake behaviour through different receptors. Currently, how these various receptors are themselves regulated is still not completely understood. The KCTD12-family of proteins, which have been shown to modify G-protein-coupled receptor (GPCR) signalling in mammals, are one possibility of auxiliary proteins modulating 5-HT receptor signalling. The KCTD12-family was found to be remarkably conserved and present in species from C. elegans to humans. The Drosophila KCTD12 homologue Kctd12-like (Ktl) is highly expressed in both the larval and adult CNS. By performing behavioural assays in male Drosophila, this study now reveasl that Ktl is required for proper male aggression and mating behaviour. Previously, it was shown that Ktl is in a complex with the Drosophila 5-HT receptor 5-HT7, and both Ktl and the 5-HT1A receptor are required in insulin-producing cells (IPCs) for proper adult male behaviour, as well as for hyperaggressive activity induced by the mammalian 5-HT1A receptor agonist 8-hydroxy-2-dipropylaminotetralin-hydrobromide. Finally, Ktl expression in the IPCs is necessary to regulate locomotion and normal sleep/wake patterns in Drosophila, but not the 5-HT1A receptor. Similar to what was observed with mammalian KCTD12-family members that interact physically with a GPCR receptor to regulate desensitization, in Drosophila Ktl may function in GPCR 5-HT receptor pathways to regulate their signalling, which is required for proper adult male behaviour.
Pilorz, V., Cunningham, P. S., Jackson, A., West, A. C., Wager, T. T., Loudon, A. S. and Bechtold, D. A. (2014). A novel mechanism controlling resetting speed of the circadian clock to environmental stimuli. Curr Biol 24: 766-773. PubMed ID: 24656826
Many aspects of mammalian physiology are driven through the coordinated action of internal circadian clocks. Clock speed (period) and phase (temporal alignment) are fundamental to an organism's ability to synchronize with its environment. In humans, lifestyles that disturb these clocks, such as shift work, increase the incidence of diseases such as cancer and diabetes. Casein kinases 1δ and ε (homologs of Drosophila Doubletime) are closely related clock components implicated in period determination. However, CK1δ is so dominant in this regard that it remains unclear what function CK1epsilon; normally serves. This study has reveal3e that CK1ε dictates how rapidly the clock is reset by environmental stimuli. Genetic disruption of CK1ε in mice enhances phase resetting of behavioral rhythms to acute light pulses and shifts in light cycle. This impact of CK1ε targeting is recapitulated in isolated brain suprachiasmatic nucleus and peripheral (lung) clocks during NMDA- or temperature-induced phase shift in association with altered PERIOD (PER; see Drosophila Period) protein dynamics. Importantly, accelerated re-entrainment of the circadian system in vivo and in vitro can be achieved in wild-type animals through pharmacological inhibition of CK1ε. These studies therefore reveal a role for CK1ε in stabilizing the circadian clock against phase shift and highlight it as a novel target for minimizing physiological disturbance in shift workers.
Konig, C., Schleyer, M., Leibiger, J., El-Keredy, A. and Gerber, B. (2014). Bitter-Sweet Processing in Larval Drosophila. Chem Senses [Epub ahead of print]. PubMed ID: 24833133
'Sweet-' and 'bitter-' tasting substances distinctively support attractive and aversive choice behavior, respectively, and therefore are thought to be processed by distinct pathways. Interestingly, electrophysiological recordings in adult Drosophila suggest that bitter and salty tastants, in addition to activating bitter, salt, or bitter/salt sensory neurons, can also inhibit sweet-sensory neurons. However, the behavioral significance of such a potential for combinatorial coding is little understood. Using larval Drosophila as a study case, this study found that the preference towards fructose is inhibited when assayed in the background of the bitter tastant quinine. When testing the influence of quinine on the preference to other, equally preferred sweet tastants, it was found that these sweet tastants differ in their susceptibility to be inhibited by quinine. Such stimulus specificity argues that the inhibitory effect of quinine is not due to general effects on locomotion or nausea. In turn, not all bitter tastants have the same potency to inhibit sweet preference; notably, their inhibitory potency is not determined by the strength of the avoidance of them. Likewise, equally avoided concentrations of sodium chloride differ in their potency to inhibit sugar preference. Furthermore, Gr33a-Gal4-positive neurons, while being necessary for bitter avoidance, are dispensable for inhibition of the sweet pathway. Thus, interactions across taste modalities are behaviorally significant and, arguably diverse in mechanism. These results suggest that the coding of tastants and the organization of gustatory behavior may be more combinatorial than is generally acknowledged.
Saturday, May 17th
Gafner, L., Dalessi, S., Escher, E., Pyrowolakis, G., Bergmann, S. and Basler, K. (2013). Manipulating the sensitivity of signal-induced repression: quantification and consequences of altered brinker gradients. PLoS One 8: e71224. PubMed ID: 23951114
Traditionally, the analysis of gene regulatory regions suffered from the caveat that it was restricted to artificial contexts (e.g. reporter constructs of limited size). With the advent of the BAC recombineering technique, genomic constructs can now be generated to test regulatory elements in their endogenous environment. The expression of the transcriptional repressor brinker (brk) is negatively regulated by Dpp signaling. Repression is mediated by small sequence motifs, the silencer elements (SEs), that are present in multiple copies in the regulatory region of brk. This study manipulated the SEs in the brk locus. The effects of the individual SEs on the Brk gradient in the wing disc were precisely quantified by employing a 1D data extraction method, followed by the quantification of the data with reference to an internal control. It was found that mutating the SEs results in an expansion of the brk expression domain. However, even after mutating all predicted SEs, repression could still be observed in regions of maximal Dpp levels. Thus, the data point to the presence of additional, low affinity binding sites in the brk locus.
Boube, M., Hudry, B., Immarigeon, C., Carrier, Y., Bernat-Fabre, S., Merabet, S., Graba, Y., Bourbon, H. M. and Cribbs, D. L. (2014). Drosophila melanogaster Hox Transcription Factors Access the RNA Polymerase II Machinery through Direct Homeodomain Binding to a Conserved Motif of Mediator Subunit Med19. PLoS Genet 10: e1004303. PubMed ID: 24786462
Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits organized in modules that bridge the PolII enzyme to DNA-bound TFs. This study investigate the physical and functional interplay between Drosophila melanogaster Hox developmental TFs and MED complex proteins. The Med19 subunit was found to directly bind Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, a role was identified for Med19 in Hox-dependent target gene activation. A conserved, animal-specific motif was found that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded 'co-factor' that acts together with Hox proteins through their homeodomains in regulated developmental transcription.
Lettice, L. A., Williamson, I., Devenney, P. S., Kilanowski, F., Dorin, J. and Hill, R. E. (2014). Development of five digits is controlled by a bipartite long-range cis-regulator. Development 141: 1715-1725. PubMed ID: 24715461
Conservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. This study examined the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator responsible for the spatiotemporal control of Shh (see Drosophila Hedgehog) in the murine limb bud. By systematically dissecting the ZRS, both in transgenic assays and within in the endogenous locus, the ZRS was shown, in effect, to be composed of two distinct domains of activity: one domain directs spatiotemporal activity but functions predominantly from a short range, whereas a second domain is required to promote long-range activity. These two domains encode activities that are highly integrated, and the second domain is crucial in promoting the chromosomal conformational changes correlated with gene activity. During limb bud development, these activities encoded by the ZRS are interpreted differently by the fore limbs and the hind limbs; in the absence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels of Shh lead to a variant digit pattern ranging from two to four digits. Hence, in the embryo, the second domain stabilises the developmental programme providing a buffer for SHH morphogen activity and this ensures that five digits form in both sets of limbs.
Palsson, A., Wesolowska, N., Reynisdottir, S., Ludwig, M. Z. and Kreitman, M. (2014). Naturally Occurring Deletions of Hunchback Binding Sites in the Even-Skipped Stripe 3+7 Enhancer. PLoS One 9: e91924. PubMed ID: 24786295
Changes in regulatory DNA contribute to phenotypic differences within and between taxa. Comparative studies show that many transcription factor binding sites (TFBS) are conserved between species whereas functional studies reveal that some mutations segregating within species alter TFBS function. Consistently, in this analysis of 13 regulatory elements in Drosophila melanogaster populations, single base and insertion/deletion polymorphism are rare in characterized regulatory elements. Experimentally defined TFBS are nearly devoid of segregating mutations and, as has been shown before, are quite conserved. For instance 8 of 11 Hunchback binding sites in the stripe 3+7 enhancer of even-skipped are conserved between D. melanogaster and Drosophila virilis. Oddly, a 72 bp deletion was found that removes one of these binding sites (Hb8), segregating within D. melanogaster. Furthermore, a 45 bp deletion polymorphism in the spacer between the stripe 3+7 and stripe 2 enhancers, removes another predicted Hunchback site. These two deletions are separated by approximately 250 bp, sit on distinct haplotypes, and segregate at appreciable frequency. The Hb8Delta is at 5 to 35% frequency in the new world, but also shows cosmopolitan distribution. There is depletion of sequence variation on the Hb8Delta-carrying haplotype. Quantitative genetic tests indicate that Hb8Delta affects developmental time, but not viability of offspring. The Eve expression pattern differs between inbred lines, but the stripe 3 and 7 boundaries seem unaffected by Hb8Delta. The data reveal segregating variation in regulatory elements, which may reflect evolutionary turnover of characterized TFBS due to drift or co-evolution.
Friday, May 16th
Binggeli, O., Neyen, C., Poidevin, M. and Lemaitre, B. (2014). Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila. PLoS Pathog 10: e1004067. PubMed ID: 24788090
The melanization reaction is a major immune response in Arthropods and involves the rapid synthesis of melanin at the site of infection and injury. A key enzyme in the melanization process is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones, which subsequently polymerize into melanin. The Drosophila genome encodes three POs, which are primarily produced as zymogens or prophenoloxidases (PPO). Two of them, PPO1 and PPO2, are produced by crystal cells. This study generated flies carrying deletions in PPO1 and PPO2. By analyzing these mutations alone and in combination, the functions were clarified of both PPOs in humoral melanization. PPO1 and PPO2 were shown to be responsible for all the PO activity in the hemolymph. While PPO1 is involved in the rapid early delivery of PO activity, PPO2 is accumulated in the crystals of crystal cells and provides a storage form that can be deployed in a later phase. This study also reveals an important role for PPO1 and PPO2 in the survival to infection with Gram-positive bacteria and fungi, underlining the importance of melanization in insect host defense.
Kuuluvainen, E., Hakala, H., Havula, E., Sahal Estime, M., Ramet, M., Hietakangas, V. and Makela, T. P. (2014). Cyclin-dependent kinase 8 module expression profiling reveals requirement of Mediator subunits 12 and 13 for transcription of Serpent-dependent innate immunity genes in Drosophila. J Biol Chem [Epub ahead of print]. PubMed ID: 24778181
The Cdk8 (cyclin-dependent kinase 8) module of Mediator integrates regulatory cues from transcription factors to RNA Polymerase II (RNAPII). It consists of four subunits where Med12 and Med13 link Cdk8 and cyclin C (CycC) to core Mediator. This study investigated the contributions of the Cdk8 module subunits to transcriptional regulation using RNA interference in Drosophila cells. Genome-wide expression profiling demonstrated separation of Cdk8-CycC and Med12-Med13 profiles. However, transcriptional regulation by Cdk8-CycC was dependent on Med12-Med13. This observation also revealed that Cdk8-CycC and Med12-Med13 often have opposite transcriptional effects. Interestingly, Med12 and Med13 profiles overlapped significantly with that of the GATA factor Serpent. Accordingly, mutational analyses indicated that GATA sites are required for Med12-Med13 regulation of Serpent-dependent genes. Med12 and Med13 were also found to be required for Serpent-activated innate immunity genes in defense to bacterial infection. The results reveal a novel role for the Cdk8 module in Serpent-dependent transcription and innate immunity.
Taillebourg, E., Schneider, D. S. and Fauvarque, M. O. (2014). The Drosophila Deubiquitinating Enzyme dUSP36 Acts in the Hemocytes for Tolerance to Listeria monocytogenes Infections. J Innate Immun. PubMed ID: 24777180
Listeria monocytogenes is a facultative intracellular pathogen which can infect Drosophila melanogaster. Upon infection, Drosophila mounts an immune response including antimicrobial peptide production and autophagy activation. A set of previously published results prompted a study of the role of the deubiquitinating enzyme dUSP36 in response to L. monocytogenes infections. Flies with dUsp36-specific inactivation in hemocytes were shown to be susceptible to L. monocytogenes infections (as are flies with autophagy-deficient hemocytes) but are still able to control bacterial growth. Interestingly, flies with dUsp36-depleted hemocytes are not sensitized to infection by other pathogens. It is concluded that dUsp36 plays a major role in hemocytes for tolerance to L. monocytogenes.
Tan, K. L., Vlisidou, I. and Wood, W. (2014). Ecdysone Mediates the Development of Immunity in the Drosophila Embryo. Curr Biol [Epub ahead of print]. 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.
Thursday, May 15th
Hetie, P., de Cuevas, M. and Matunis, E. (2014). Conversion of Quiescent Niche Cells to Somatic Stem Cells Causes Ectopic Niche Formation in the Drosophila Testis. Cell Rep [Epub ahead of print]. PubMed ID: 24746819
Adult stem cells reside in specialized regulatory microenvironments, or niches, where local signals ensure stem cell maintenance. The Drosophila testis contains a well-characterized niche wherein signals from postmitotic hub cells promote maintenance of adjacent germline stem cells and somatic cyst stem cells (CySCs). Hub cells were considered to be terminally differentiated; this study shows that they can give rise to CySCs. Genetic ablation of CySCs triggers hub cells to transiently exit quiescence, delaminate from the hub, and convert into functional CySCs. Ectopic Cyclin D-Cdk4 expression in hub cells is also sufficient to trigger their conversion into CySCs. In both cases, this conversion causes the formation of multiple ectopic niches over time. Therefore, this work provides a model for understanding how oncogenic mutations in quiescent niche cells could promote loss of quiescence, changes in cell fate, and aberrant niche expansion.
Voog, J., Sandall, S. L., Hime, G. R., Resende, L. P., Loza-Coll, M., Aslanian, A., Yates, J. R., 3rd, Hunter, T., Fuller, M. T. and Jones, D. L. (2014). Escargot Restricts Niche Cell to Stem Cell Conversion in the Drosophila Testis. Cell Rep [Epub ahead of print]. PubMed ID: 24794442
Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. Esg-interacting proteins were identified, and an interaction was demonstrated between Esg and the corepressor C-terminal binding protein (CtBP), which is also required for maintenance of hub cell fate. These results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo.
Huang, J. and Kalderon, D. (2014). Coupling of Hedgehog and Hippo pathways promotes stem cell maintenance by stimulating proliferation. J Cell Biol [Epub ahead of print]. PubMed ID: 24798736
It is essential to define the mechanisms by which external signals regulate adult stem cell numbers, stem cell maintenance, and stem cell proliferation to guide regenerative stem cell therapies and to understand better how cancers originate in stem cells. This paper shows that Hedgehog (Hh) signaling in Drosophila melanogaster ovarian follicle stem cells (FSCs) induces the activity of Yorkie (Yki), the transcriptional coactivator of the Hippo pathway, by inducing yki transcription. Moreover, both Hh signaling and Yki positively regulate the rate of FSC proliferation, both are essential for FSC maintenance, and both promote increased FSC longevity and FSC duplication when in excess. It was also found that responses to activated Yki depend on Cyclin E induction while responses to excess Hh signaling depend on Yki induction, and excess Yki can compensate for defective Hh signaling. These causal connections provide the most rigorous evidence to date that a niche signal can promote stem cell maintenance principally by stimulating stem cell proliferation.
Berns, N., Woichansky, I., Friedrichsen, S., Kraft, N. and Riechmann, V. (2014). A genome-scale in vivo RNAi analysis of epithelial development in Drosophila identifies new proliferation domains outside of the stem cell niche. J Cell Sci [Epub ahead of print]. PubMed ID: 24762813
The Drosophila oogenesis system provides an excellent model to study the development of epithelial tissues. This study reports the first genome-scale in vivo RNAi screen for genes controlling epithelial development. By directly analysing cell and tissue architecture, 1125 genes were identified that were assigned to seven different functions in epithelial formation and homeostasis. The significance of the screen was validated by generating mutants for Vps60, a component of the ESCRT machinery. This analysis provided new insights into spatiotemporal control of cell proliferation in the follicular epithelium. Previous studies identified signals controlling divisions in the follicle stem cell niche. However, 99% of cell divisions occur outside of the niche and it is unclear how these divisions are controlled. The data distinguish two new domains with differential proliferation control outside of the stem cell niche. One domain abuts the niche and is characterised by ESCRT, Notch and JAK/STAT mediated proliferation control. Adjacently, another domain is defined by loss of ESCRT impact on cell division. Thus, during development epithelial cells pass through different modes of proliferation control. The switch between these modes might reflect regressing stemness of epithelial cells over time.
Updike, D. L., Knutson, A. K., Egelhofer, T. A., Campbell, A. C. and Strome, S. (2014). Germ-Granule Components Prevent Somatic Development in the C. elegans Germline. Curr Biol 24: 970-975. PubMed ID: 24746798
Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans. In Drosophila, germ granules have been implicated in germline determination. C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination, but their components are still needed for fertility. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g., in germlines lacking MEX-3 and GLD-1 (Drosophila homolog: Held out wings) or upon neuronal induction by CHE-1 (Drosophila homolog: Glass). However, it has not been established whether loss of P granules is the cause or effect of cell fate transformation. To test cause and effect, P granules were severly compromised by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization [GLH-1 (see Drosophila Vasa) and GLH-4] and an investigation was carried out to see whether this causes germ cells to lose totipotency and initiate somatic reprogramming. It was found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate.
Wednesday, May 14th
Mirth, C. K., Tang, H. Y., Makohon-Moore, S. C., Salhadar, S., Gokhale, R. H., Warner, R. D., Koyama, T., Riddiford, L. M. and Shingleton, A. W. (2014). Juvenile hormone regulates body size and perturbs insulin signaling in Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 24778227
The role of juvenile hormone (JH; see Metamorphosis and development of the adult fly) in regulating the timing and nature of insect molts is well-established. Increasing evidence suggests that JH is also involved in regulating final insect size. This study has elucidatex the developmental mechanism through which JH regulates body size in developing Drosophila larvae by genetically ablating the JH-producing organ, the corpora allata (CA). Larvae that lack CA were found to pupariate at smaller sizes than control larvae due to a reduced larval growth rate. Neither the timing of the metamorphic molt nor the duration of larval growth was affected by the loss of JH. Further, it was shown that the effects of JH on growth rate are dependent on the forkhead box O transcription factor (FOXO), which is negatively regulated by the insulin-signaling pathway. Larvae that lacked the CA had elevated levels of FOXO activity, whereas a loss-of-function mutation of FOXO rescued the effects of CA ablation on final body size. Finally, the effect of JH on growth appears to be mediated, at least in part, via ecdysone synthesis in the prothoracic gland. These results indicate a role of JH in regulating growth rate via the ecdysone- and insulin-signaling pathways.
Perez-Moreno, J. J., Bischoff, M., Martin-Bermudo, M. D. and Estrada, B. (2014). The conserved transmembrane proteoglycan Perdido/Kon-tiki is essential for myofibrillogenesis and sarcomeric structure in Drosophila. J Cell Sci [Epub ahead of print]. PubMed ID: 24794494
Muscle differentiation requires the assembly of high-order structures called myofibrils, composed of sarcomeres. Even though the molecular organization of sarcomeres is well known, the mechanisms underlying myofibrillogenesis are poorly understood. It has been proposed that integrin-dependent adhesion nucleates myofibril at the periphery of the muscle cell to sustain sarcomere assembly. This study reports a role for the gene perdido (perd, also known as kon-tiki, a transmembrane chondroitin proteoglycan) in myofibrillogenesis. Expression of perd RNAi in muscles, prior to adult myogenesis, can induce misorientation and detachment of Drosophila adult abdominal muscles. In comparison to controls, perd-depleted muscles contain fewer myofibrils, localized at the cell periphery. These myofibrils are detached from each other and display a defective sarcomeric structure. These results demonstrate that the extracellular matrix receptor Perd has a specific role in the assembly of myofibrils and in sarcomeric organization. It is suggested that Perd acts downstream or in parallel to integrins to enable the connection of nascent myofibrils to the Z-bands. This work identifies the Drosophila adult abdominal muscles as a model to investigate in vivo the mechanisms behind myofibrillogenesis.
Dong, B., Hannezo, E. and Hayashi, S. (2014). Balance between Apical Membrane Growth and Luminal Matrix Resistance Determines Epithelial Tubule Shape. Cell Rep [Epub ahead of print]. PubMed ID: 24794438
The morphological stability of biological tubes is crucial for the efficient circulation of fluids and gases. Failure of this stability causes irregularly shaped tubes found in multiple pathological conditions. This study reports that Drosophila mutants of the ESCRT III component Shrub/Vps32 exhibit a strikingly elongated sinusoidal tube phenotype. This is caused by excessive apical membrane synthesis accompanied by the ectopic accumulation and overactivation of Crumbs in swollen endosomes. Furthermore, it was demonstrated that the apical extracellular matrix (aECM) of the tracheal tube is a viscoelastic material coupled with the apical membrane. A simple mechanical model is presented in which aECM elasticity, apical membrane growth, and their interaction are three vital parameters determining the stability of biological tubes. The findings demonstrate a mechanical role for the extracellular matrix and suggest that the interaction of the apical membrane and an elastic aECM determines the final morphology of biological tubes independent of cell shape.
Averbukh, I., Ben-Zvi, D., Mishra, S. and Barkai, N. (2014). Scaling morphogen gradients during tissue growth by a cell division rule. Development 141: 2150-2156. PubMed ID: 24803660
Morphogen gradients guide the patterning of tissues and organs during the development of multicellular organisms. In many cases, morphogen signaling is also required for tissue growth. The consequences of this interplay between growth and patterning are not well understood. In the Drosophila wing imaginal disc, the morphogen Dpp guides patterning and is also required for tissue growth. In particular, it was recently reported that cell division in the disc correlates with the temporal increase in Dpp signaling. This study mathematically modeled morphogen gradient formation in a growing tissue, accounting also for morphogen advection and dilution. The analysis defines a new scaling mechanism, which was termed the morphogen-dependent division rule (MDDR): when cell division depends on the temporal increase in morphogen signaling, the morphogen gradient scales with the growing tissue size, tissue growth becomes spatially uniform and the tissue naturally attains a finite size. This model is consistent with many properties of the wing disc. However, it was found that the MDDR is not consistent with the phenotype of scaling-defective mutants, supporting the view that temporal increase in Dpp signaling is not the driver of cell division during late phases of disc development. More generally, the results show that local coupling of cell division with morphogen signaling can lead to gradient scaling and uniform growth even in the absence of global feedbacks. The MDDR scaling mechanism might be particularly beneficial during rapid proliferation, when global feedbacks are hard to implement.
Gummalla, M., Galetti, S., Maeda, R. K. and Karch, F. (2014). Hox gene regulation in the central nervous system of Drosophila. Front Cell Neurosci 8: 96. PubMed ID: 24795565
Hox genes specify the structures that form along the anteroposterior (AP) axis of bilateria. Within the genome, they often form clusters where, remarkably enough, their position within the clusters reflects the relative positions of the structures they specify along the AP axis. This correspondence between genomic organization and gene expression pattern has been conserved through evolution and provides a unique opportunity to study how chromosomal context affects gene regulation. In Drosophila, a general rule, often called “posterior dominance,” states that Hox genes specifying more posterior structures repress the expression of more anterior Hox genes. This rule explains the apparent spatial complementarity of Hox gene expression patterns in Drosophila. This paper reviews a noticeable exception to this rule where the more-posteriorly expressed Abd-B Hox gene fails to repress the more-anterior abd-A gene in cells of the central nervous system (CNS). While Abd-B is required to repress ectopic expression of abd-A in the posterior epidermis, abd-A repression in the posterior CNS is accomplished by a different mechanism that involves a large 92 kb long non-coding RNA (lncRNA) encoded by the intergenic region separating abd-A and Abd-B (the iab8ncRNA). Dissection of this lncRNA revealed that abd-A is repressed by the lncRNA using two redundant mechanisms. The first mechanism is mediated by a microRNA (mir-iab-8) encoded by intronic sequence within the large iab8-ncRNA. Meanwhile, the second mechanism seems to involve transcriptional interference by the long iab-8 ncRNA on the abd-A promoter. Recent work demonstrating CNS-specific regulation of genes by ncRNAs in Drosophila, seem to highlight a potential role for the iab-8-ncRNA in the evolution of the Drosophila Hox complexes.
Tuesday, May 13th
Fu, S., Nien, C. Y., Liang, H. L. and Rushlow, C. (2014). Co-activation of microRNAs by Zelda is essential for early Drosophila development. Development. PubMed ID: 24764079
Transcription factors and microRNAs (miRNAs) are two important classes of trans-regulators in differential gene expression. Transcription factors occupy cis-regulatory motifs in DNA to activate or repress gene transcription, whereas miRNAs specifically pair with seed sites in target mRNAs to trigger mRNA decay or inhibit translation. Dynamic spatiotemporal expression patterns of transcription factors and miRNAs during development point to their stage- and tissue-specific functions. Recent studies have focused on miRNA functions during development; however, much remains to explore regarding how the expression of miRNAs is initiated and how dynamic miRNA expression patterns are achieved by transcriptional regulatory networks at different developmental stages. This study focused on the identification, regulation and function of miRNAs during the earliest stage of Drosophila development, when the maternal-to-zygotic transition (MZT) takes place. Eleven miRNA clusters comprise the first set of miRNAs activated in the blastoderm embryo. The transcriptional activator Zelda is required for their proper activation and regulation, and Zelda binding observed in genome-wide binding profiles is predictive of enhancer activity. In addition, other blastoderm transcription factors, comprising both activators and repressors, the activities of which are potentiated and coordinated by Zelda, contribute to the accurate temporal and spatial expression of these miRNAs, which are known to function in diverse developmental processes. Although previous genetic studies showed no early phenotypes upon loss of individual miRNAs, this analysis of the miR-1; miR-9a double mutant revealed defects in gastrulation, demonstrating the importance of co-activation of miRNAs by Zelda during the MZT.
Hatori, R., Ando, T., Sasamura, T., Nakazawa, N., Nakamura, M., Taniguchi, K., Hozumi, S., Kikuta, J., Ishii, M. and Matsuno, K. (2014). Left-right asymmetry is formed in individual cells by intrinsic cell chirality. Mech Dev. PubMed ID: 24800645
Many animals show left-right (LR) asymmetric morphology. The mechanisms of LR asymmetric development are evolutionarily divergent, and they remain elusive in invertebrates. Various organs in Drosophila melanogaster show stereotypic LR asymmetry, including the embryonic gut. The Drosophila embryonic hindgut twists 90 degrees left-handedly, thereby generating directional LR asymmetry. It has been recently revealed that the hindgut epithelial cell is chiral in shape and in other properties; this is termed planar cell chirality (PCC). It has been previously shown by computer modeling that PCC is sufficient to induce the hindgut rotation. In addition, both the PCC and the direction of hindgut twisting are reversed in Myosin31DF (Myo31DF) mutants. Myo31DF encodes Drosophila MyosinID, an Actin-based motor protein, whose molecular functions in LR asymmetric development are largely unknown. To understand how PCC directs the asymmetric cell-shape, this study analyzed PCC in genetic mosaics composed of cells homozygous for mutant Myo31DF, some of which also overexpressed wild-type Myo31DF. Wild-type cell-shape chirality only formed in the Myo31DF-overexpressing cells, suggesting that cell-shape chirality was established in each cell and reflects intrinsic PCC. A computer model recapitulating the development of this genetic mosaic suggested that mechanical interactions between cells are required for the cell-shape behavior seen in vivo. Mosaic analysis also suggested that during hindgut rotation in vivo, wild-type Myo31DF suppresses the elongation of cell boundaries, supporting the idea that cell-shape chirality is an intrinsic property determined in each cell. However, the amount and distribution of F-actin and Myosin II, which are known to help generate the contraction force on cell boundaries, did not show differences between Myo31DF mutant cells and wild-type cells, suggesting that the static amount and distribution of these proteins are not involved in the suppression of cell-boundary elongation. Taken together, these results suggest that cell-shape chirality is intrinsically formed in each cell, and that mechanical force from intercellular interactions contributes to its formation and/or maintenance.
Reversi, A., Loeser, E., Subramanian, D., Schultz, C. and De Renzis, S. (2014). Plasma membrane phosphoinositide balance regulates cell shape during Drosophila embryo morphogenesis. J Cell Biol. PubMed ID: 24798734
Remodeling of cell shape during morphogenesis is driven by the coordinated expansion and contraction of specific plasma membrane domains. Loss of this coordination results in abnormal cell shape and embryonic lethality. This study shows that plasma membrane lipid composition plays a key role in coordinating plasma membrane contraction during expansion. An increase in PI(4,5)P2 levels was found to cause premature actomyosin contraction, resulting in the formation of shortened cells. Conversely, acute depletion of PI(4,5)P2 blocked plasma membrane expansion and led to premature actomyosin disassembly. PI(4,5)P2-mediated contractility is counteracted by PI(3,4,5)P3 and the zygotic gene bottleneck, which acts by limiting myosin recruitment during plasma membrane expansion. Collectively, these data support a model in which the ratio of PI(4,5)P2/PI(3,4,5)P3 coordinates actomyosin contractility and plasma membrane expansion during tissue morphogenesis, thus ensuring proper cell shape.
Radermacher, P. T., Myachina, F., Bosshardt, F., Pandey, R., Mariappa, D., Muller, H. A. and Lehner, C. F. (2014). O-GlcNAc reports ambient temperature and confers heat resistance on ectotherm development. Proc Natl Acad Sci U S A. PubMed ID: 24706800
Effects of temperature on biological processes are complex. Diffusion is less affected than the diverse enzymatic reactions that have distinct individual temperature profiles. Hence thermal fluctuations pose a formidable challenge to ectothermic organisms in which body temperature is largely dictated by the ambient temperature. How cells in ectotherms cope with the myriad disruptive effects of temperature variation is poorly understood at the molecular level. This study shows that nucleocytoplasmic posttranslational modification of proteins with O-linked GlcNAc (O-GlcNAc) is closely correlated with ambient temperature during development of distantly related ectotherms ranging from the insect Drosophila melanogaster to the nematode Caenorhabditis elegans to the fish Danio rerio. Regulation seems to occur at the level of activity of the only two enzymes, O-GlcNAc transferase and O-GlcNAcase, that add and remove, respectively, this posttranslational modification in nucleus and cytoplasm. With genetic approaches in D. melanogaster and C. elegans, this study demonstrates the importance of high levels of this posttranslational modification for successful development at elevated temperatures. Because many cytoplasmic and nuclear proteins in diverse pathways are O-GlcNAc targets, temperature-dependent regulation of this modification might contribute to an efficient coordinate adjustment of cellular processes in response to thermal change.
Monday, May 12th
Sechi, S., Colotti, G., Belloni, G., Mattei, V., Frappaolo, A., Raffa, G. D., Fuller, M. T. and Giansanti, M. G. (2014). GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster. PLoS Genet 10: e1004305. PubMed ID: 24786584
The highly conserved Golgi phosphoprotein 3 (GOLPH3) protein has been described as a Phosphatidylinositol 4-phosphate [PI(4)P] effector at the Golgi. GOLPH3 is also known as a potent oncogene, commonly amplified in several human tumors. However, the molecular pathways through which the oncoprotein GOLPH3 acts in malignant transformation are largely unknown. GOLPH3 has never been involved in cytokinesis. This study characterizes the Drosophila melanogaster homologue of human GOLPH3 during cell division. GOLPH3/Rotini was shown to accumulate at the cleavage furrow and is required for successful cytokinesis in Drosophila spermatocytes and larval neuroblasts. In premeiotic spermatocytes GOLPH3 protein is required for maintaining the organization of Golgi stacks. In dividing spermatocytes GOLPH3 is essential for both contractile ring and central spindle formation during cytokinesis. Wild type function of GOLPH3 enables maintenance of centralspindlin and Rho1 at cell equator and stabilization of Myosin II and Septin rings. The molecular mechanism underlying GOLPH3 function in cytokinesis is strictly dependent on the ability of this protein to interact with PI(4)P. Mutations that abolish PI(4)P binding impair recruitment of GOLPH3 to both the Golgi and the cleavage furrow. Moreover telophase cells from mutants with defective GOLPH3-PI(4)P interaction fail to accumulate PI(4)P-and Rab11-associated secretory organelles at the cleavage site. Finally, it was shown that GOLPH3 protein interacts with components of both cytokinesis and membrane trafficking machineries in Drosophila cells. Based on these results it is proposed that GOLPH3 acts as a key molecule to coordinate phosphoinositide signaling with actomyosin dynamics and vesicle trafficking during cytokinesis. Because cytokinesis failures have been associated with premalignant disease and cancer, these studies suggest novel insight into molecular circuits involving the oncogene GOLPH3 in cytokinesis.
Riparbelli, M. G., Gottardo, M., Glover, D. M. and Callaini, G. (2014). Inhibition of Polo kinase by BI2536 affects centriole separation during Drosophila male meiosis. Cell Cycle 13 [Epub ahead of print]. PubMed ID: 24802643
Pharmacological inhibition of Drosophila Polo kinase with BI2536 has allowed reexamination of the requirements for Polo during Drosophila male gametogenesis. BI2536-treated spermatocytes persist in a pro-metaphase state without dividing and have condensed chromosomes that do not separate. Centrosomes failed to recruit γ-tubulin and centrosomin (Cnn) and are not associated with microtubule arrays that are abnormal and do not form proper bipolar spindles. Centrioles, which usually separate during the anaphase of the first meiosis, remain held together in a V-shaped configuration suggesting that Polo kinase regulates the proteolysis that breaks centriole linkage to ensure their disengagement. Despite these defects spermatid differentiation proceeds, leading to axoneme formation.
Kitagawa, M., Fung, S. Y., Hameed, U. F., Goto, H., Inagaki, M. and Lee, S. H. (2014). Cdk1 Coordinates Timely Activation of MKlp2 Kinesin with Relocation of the Chromosome Passenger Complex for Cytokinesis. Cell Rep 7: 166-179. PubMed ID: 24656812
The chromosome passenger complex (CPC) must relocate from anaphase chromosomes to the cell equator for successful cytokinesis during mitosis. Although this landmark event requires the mitotic kinesin MKlp2 (Drosophila homolog: Subito), the spatiotemporal mechanistic basis remains elusive. This study shows that phosphoregulation of MKlp2 by the mitotic kinase Cdk1/cyclin B1 (see Drosophila Cyclin B and Cdc2) coordinates proper mitotic transition with CPC relocation. Multiple Cdk1/cyclin B1 phosphorylation sites were identified within the stalk and C-terminal tail that inhibit microtubule binding and bundling, oligomerization/clustering, and chromosome targeting of MKlp2. Specifically, inhibition of these abilities by Cdk1/cyclin B1 phosphorylation is essential for proper early mitotic progression. Upon anaphase onset, however, reversal of Cdk1/cyclin B1 phosphorylation promotes MKlp2-CPC complex formation and relocates the CPC from anaphase chromosomes for successful cytokinesis. Thus, it is proposed that phosphoregulation of MKlp2 by Cdk1/cyclin B1 ensures that activation of MKlp2 kinesin and relocation of the CPC occur at the appropriate time and space for proper mitotic progression and genomic stability.
Rattani, A., Vinod, P. K., Godwin, J., Tachibana-Konwalski, K., Wolna, M., Malumbres, M., Novak, B. and Nasmyth, K. (2014). Dependency of the spindle assembly checkpoint on Cdk1 renders the anaphase transition irreversible. Curr Biol 24: 630-637. PubMed ID: 24583015
Activation of anaphase-promoting complex/cyclosome (APC/CCdc20) by Cdc20 (see Drosophila Fizzy) is delayed by the spindle assembly checkpoint (SAC). When all kinetochores come under tension, the SAC is turned off and APC/CCdc20 degrades cyclin B (see Drosophila Cyclin B) and securin, which activates separase (see Drosophila Separase). The latter then cleaves cohesin holding sister chromatids together. Because cohesin cleavage also destroys the tension responsible for turning off the SAC, cells must possess a mechanism to prevent SAC reactivation during anaphase, which could be conferred by a dependence of the SAC on Cdk1 (see Drosophila Cdc2. To test this, mouse oocytes and embryos expressing nondegradable cyclin B were analyzed together with a Cdk1-resistant form of separase. After biorientation and SAC inactivation, APC/CCdc20 activates separase but the resulting loss of (some) cohesion is accompanied by SAC reactivation and APC/CCdc20 inhibition, which aborts the process of further securin degradation. Cyclin B is therefore the only APC/CCdc20 substrate whose degradation at the onset of anaphase is necessary to prevent SAC reactivation. The mutual activation of tension sensitive SAC and Cdk1 creates a bistable system that ensures complete activation of separase and total downregulation of Cdk1 when all chromosomes have bioriented.
Sunday, May 11th
Chin, A. L., Lin, C. Y., Fu, T. F., Dickson, B. J. and Chiang, A. S. (2014). Diversity and wiring variability of visual local neurons in the Drosophila medulla M6 stratum. J Comp Neurol. PubMed ID: 24782245
Local neurons in the vertebrate retina are instrumental in transforming visual inputs to extract contrast, motion, and color information, and in shaping bipolar-to-ganglion cell transmission to the brain. In Drosophila, UV vision is represented by R7 inner photoreceptor neurons of the eye that project to optic lobe the medulla M6 stratum, with relatively little being known of this downstream substrate. In this study, using R7 terminals as references, a 3D volume model of the M6 stratum was generated which revealed a retinotopic map for UV representations. Using this volume model as a common 3D framework, the spatial distributions of more than 200 single M6-specific local neurons (M6-LNs) were compiled and analyzed. Based on the segregation of putative dendrites and axons, these local neurons were classified into two families: directional and non-directional. Neurotransmitter immunostaining suggested a signal routing model whereby some visual information is relayed by directional M6-LNs from the anterior to the posterior M6 and all visual information is inhibited by a diverse population of non-directional M6-LNs covering the entire M6 stratum. These findings suggest that the Drosophila medulla M6 stratum contains diverse LNs that form repeating functional modules similar to those found in the vertebrate inner plexiform layer.
Montagne, C. and Gonzalez-Gaitan, M. (2014). Sara endosomes and the asymmetric division of intestinal stem cells. Development 141: 2014-2023. PubMed ID: 24803650
Tissue homeostasis is maintained by adult stem cells, which self-renew and give rise to differentiating cells. The generation of daughter cells with different fates is mediated by signalling molecules coming from an external niche or being asymmetrically dispatched between the two daughters upon stem cell mitosis. In the adult Drosophila midgut, the intestinal stem cell (ISC) divides to generate a new ISC and an enteroblast (EB) differentiating daughter. Notch signalling activity restricted to the EB regulates intestinal cell fate decision. This study shows that ISCs divide asymmetrically, and Sara endosomes in ISCs are specifically dispatched to the presumptive EB. During ISC mitosis, Notch and Delta traffic through Sara endosomes, thereby contributing to Notch signalling bias, as revealed in Sara mutants: Sara itself contributes to the control of the ISC asymmetric division. These data uncover an intrinsic endosomal mechanism during ISC mitosis, which participates in the maintenance of the adult intestinal lineage.
Zhang, L., Syed, Z. A., van Dijk Hard, I., Lim, J. M., Wells, L. and Ten Hagen, K. G. (2014). O-Glycosylation regulates polarized secretion by modulating Tango1 stability. Proc Natl Acad Sci U S A. PubMed ID: 24799692
Polarized secretion is crucial in many tissues. The conserved protein modification, O-glycosylation, plays a role in regulating secretion. However, the mechanisms by which this occurs are unknown. This study demonstrates that an O-glycosyltransferase functions as a novel regulator of secretion and secretory vesicle formation in vivo by glycosylating the essential Golgi/endoplasmic reticulum protein, Tango1 (Transport and Golgi organization 1), and conferring protection from furin-mediated proteolysis. Loss of the O-glycosyltransferase PGANT4 resulted in Tango1 cleavage, loss of secretory granules, and disrupted apical secretion. The secretory defects seen upon loss of pgant4 could be rescued either by overexpression of Tango1 or by knockdown of a specific furin (Dfur2) in vivo. These studies elucidate a novel regulatory mechanism whereby secretion is influenced by the yin/yang of O-glycosylation and proteolytic cleavage. Moreover, the data have broader implications for the potential treatment of diseases resulting from the loss of O-glycosylation by modulating the activity of specific proteases.
Rosenbaum, E. E., Vasiljevic, E., Brehm, K. S. and Colley, N. J. (2014). Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in Drosophila melanogaster. PLoS Genet 10: e1004349. PubMed ID: 24785692
As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, mutant alleles were characterized of four Drosophila glycosyl hydrolases, namely alpha-mannosidase-II (alpha-Man-II), alpha-mannosidase-IIb (alpha-Man-IIb), a beta-N-acetylglucosaminidase called Fused lobes (Fdl), and hexosaminidase 1 (Hexo1). These four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. The results reveal that alpha-Man-II and alpha-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, the results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. This study presents a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, these results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the functions of glycosyl hydrolases in the secretory pathway.
Saturday May 10th
Kasprowicz, J., Kuenen, S., Swerts, J., Miskiewicz, K. and Verstreken, P. (2014). Dynamin photoinactivation blocks Clathrin and alpha-adaptin recruitment and induces bulk membrane retrieval. J Cell Biol [Epub ahead of print]. PubMed ID: 24662566
Dynamin is a well-known regulator of synaptic endocytosis. Temperature-sensitive dynamin (shits1) mutations in Drosophila melanogaster or deletion of some of the mammalian Dynamins causes the accumulation of invaginated endocytic pits at synapses, sometimes also on bulk endosomes, indicating impaired membrane scission. However, complete loss of dynamin function has not been studied in neurons in vivo, and whether Dynamin acts in different aspects of synaptic vesicle formation remains enigmatic. This study used acute photoinactivation and found that loss of Dynamin function blocks membrane recycling and causes the buildup of huge membrane-connected cisternae, in contrast to the invaginated pits that accumulate in shits1 mutants. Moreover, photoinactivation of Dynamin in shits1 animals converts these pits into bulk cisternae. Bulk membrane retrieval has also been seen upon Clathrin photoinactivation, and superresolution imaging indicates that acute Dynamin photoinactivation blocks Clathrin and α-adaptin relocalization to synaptic membranes upon nerve stimulation. Hence, these data indicate that Dynamin is critically involved in the stabilization of Clathrin- and AP2-dependent endocytic pits.
Khadilkar, R. J., Rodrigues, D., Mote, R. D., Sinha, A. R., Kulkarni, V., Magadi, S. S. and Inamdar, M. S. (2014). ARF1-GTP regulates Asrij to provide endocytic control of Drosophila blood cell homeostasis. Proc Natl Acad Sci U S A 111: 4898-4903. PubMed ID: 24707047
Drosophila melanogaster larval hematopoiesis is a well-established model to study mechanisms that regulate hematopoietic niche maintenance and control of blood cell precursor (prohemocyte) differentiation. Molecules that perturb niche function affect the balance between prohemocytes and differentiated hemocytes. The conserved hemocyte-specific endosomal protein Asrij is essential for niche function and prohemocyte maintenance. Elucidating how subcellular trafficking molecules can regulate signaling presents an important challenge. This study shows that Asrij function is mediated by the Ras family GTPase Arf79F, the Drosophila homolog of ADP ribosylation factor 1 (ARF1), essential for clathrin coat assembly, Golgi architecture, and vesicular trafficking. Drosophila ARF1 is expressed in the larval lymph gland and in circulating hemocytes and interacts with Asrij. ARF1-depleted lymph glands show loss of niche cells and prohemocyte maintenance with increased differentiation. Inhibiting ARF1 activation by knocking down its guanine nucleotide exchange factor (Gartenzwerg) or overexpressing its GTPAse-activating protein showed that ARF1-GTP is essential for regulating niche size and maintaining stemness. Activated ARF1 regulates Asrij levels in blood cells thereby mediating Asrij function. Asrij controls crystal cell differentiation by affecting Notch trafficking. ARF1 perturbation also leads to aberrant Notch trafficking and the Notch intracellular domain is stalled in sorting endosomes. Thus, ARF1 can regulate Drosophila blood cell homeostasis by regulating Asrij endocytic function. ARF1 also regulates signals arising from the niche and differentiated cells by integrating the insulin-mediated and PDGF-VEGF receptor signaling pathways. It is proposed that the conserved ARF1-Asrij endocytic axis modulates signals that govern hematopoietic development. Thus, Asrij affords tissue-specific control of global mechanisms involved in molecular traffic.
Weinberg, J. S. and Drubin, D. G. (2014). Regulation of clathrin-mediated endocytosis by dynamic ubiquitination and deubiquitination. Curr Biol 24: 951-959. PubMed ID: 24746795
Clathrin-mediated endocytosis in budding yeast requires the regulated recruitment and disassociation of more than 60 proteins at discrete plasma membrane punctae. Posttranslational modifications such as ubiquitination may play important regulatory roles in this highly processive and ordered process. However, although ubiquitination plays an important role in cargo selection, functions for ubiquitination of the endocytic machinery are not known. This study identified the deubiquitinase (DUB) Ubp7 as a late-arriving endocytic protein. Deletion of the DUBs Ubp2 and Ubp7 resulted in elongation of endocytic coat protein lifetimes at the plasma membrane and recruitment of endocytic proteins to internal membranes. These phenotypes could be replicated by expressing a permanently ubiquitinated version of Ede1, the yeast Eps15 homolog, which is implicated in endocytic site initiation, whereas EDE1 deletion partially suppresses the DUB deletion phenotype. Both DUBs are capable of deubiquitinating Ede1 in vitro. It is concluded that deubiquitination regulates formation of endocytic sites and stability of the endocytic coat. This regulation appears to occur through Ede1, because permanently ubiquitinated Ede1 phenocopies deletion of UBP2 and UBP7. Moreover, incomplete suppression of the ubp2Δ ubp7Δ phenotype by ede1Δ indicates that ubiquitination and deubiquitination are likely to regulate additional components of the endocytic machinery.
Tognon, E., Wollscheid, N., Cortese, K., Tacchetti, C. and Vaccari, T. (2014). ESCRT-0 Is Not Required for Ectopic Notch Activation and Tumor Suppression in Drosophila. PLoS One 9: e93987. PubMed ID: 24718108
Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. This study reports that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, these in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis.
Friday, May 9th
Subtelny, A. O., Eichhorn, S. W., Chen, G. R., Sive, H. and Bartel, D. P. (2014). Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature 508: 66-71. PubMed ID: 24476825
Poly(A) tails function in transcriptional control by enhancing the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. This study describes poly(A)-tail length profiling by sequencing (PAL-seq) and applies it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other 'housekeeping' proteins tending to have shorter tails. As expected, tail lengths were coupled to translational efficiencies in early zebrafish and frog embryos. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, indicating a rapid developmental switch in the nature of translational control. This switch complements an earlier switch to zygotic transcriptional control and explains why the predominant effect of microRNA-mediated deadenylation concurrently shifts from translational repression to mRNA destabilization.
Phillips, C. M., Montgomery, B. E., Breen, P. C., Roovers, E. F., Rim, Y. S., Ohsumi, T. K., Newman, M. A., van Wolfswinkel, J. C., Ketting, R. F., Ruvkun, G. and Montgomery, T. A. (2014). MUT-14 and SMUT-1 DEAD Box RNA Helicases Have Overlapping Roles in Germline RNAi and Endogenous siRNA Formation. Curr Biol 24: 839-844. PubMed ID: 24684932
More than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized small interfering RNA (siRNA) amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells. This study shows that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNAi. Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. The targets of mut-14 and smut-1 largely overlap with the targets of other mutator class genes; however, the mut-14 smut-1 double mutant and the mut-16 mutant display the most dramatic depletion of siRNAs, suggesting that they act at a similarly early step in siRNA formation. mut-14 and smut-1 are predominantly expressed in the germline and, unlike other mutator class genes, are specifically required for RNAi targeting germline genes. A catalytically inactive, dominant-negative missense mutant of mut-14 is RNAi defective in vivo; however, mutator complexes containing the mutant protein retain the ability to synthesize siRNAs in vitro. The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs.
Shirayama, M., Stanney, W., Gu, W., Seth, M. and Mello, C. C. (2014). The Vasa Homolog RDE-12 Engages Target mRNA and Multiple Argonaute Proteins to Promote RNAi in C. elegans. Curr Biol 24: 845-851. PubMed ID: 24684931
Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi, was immunoprecipitated. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs [RDE-1 (see Drosophila Ago1) and ERGO-1], where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing.
Yang, H., Vallandingham, J., Shiu, P., Li, H., Hunter, C. P. and Mak, H. Y. (2014). The DEAD Box Helicase RDE-12 Promotes Amplification of RNAi in Cytoplasmic Foci in C. elegans. Curr Biol 24: 832-838. PubMed ID: 24684930
RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer (see Drosophila Dicer 1)and RDE-1/Argonaute (Drosophila homolog Ago1) into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. This study reports that RDE-12, a conserved a conserved Vasa-related phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. The results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments.
Fukunaga, R. and Zamore, P. D. (2014). A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis. Cell Cycle 13 [Epub ahead of print]. PubMed ID: 24787225
The enzyme Dicer (see Drosophila Dicer 1) is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? This paper reviews recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5'-monophosphate of a substrate RNA and inorganic phosphate. It is proposed that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme.
Thursday, May 8th
Tie, F., Banerjee, R., Saiakhova, A. R., Howard, B., Monteith, K. E., Scacheri, P. C., Cosgrove, M. S. and Harte, P. J. (2014). Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing. Development 141: 1129-1139. PubMed ID: 24550119
Trithorax (Trx) antagonizes epigenetic silencing by Polycomb group (PcG) proteins, stimulates enhancer-dependent transcription, and establishes a 'cellular memory' of active transcription of PcG-regulated genes. The mechanisms underlying these Trx functions remain largely unknown, but are presumed to involve its histone H3K4 methyltransferase activity. This study report that the SET domains of Trx and Trx-related (Trr) have robust histone H3K4 monomethyltransferase activity in vitro and that Tyr3701 of Trx and Tyr2404 of Trr prevent them from being trimethyltransferases. The trxZ11 missense mutation (G3601S), which abolishes H3K4 methyltransferase activity in vitro, reduces the H3 H3K4me1 but not the H3K4me3 level in vivo. trxZ11 also suppresses the impaired silencing phenotypes of the Pc3 mutant, suggesting that H3K4me1 is involved in antagonizing Polycomb silencing. Polycomb silencing is also antagonized by Trx-dependent H3K27 acetylation by CREB-binding protein (CBP). Perturbation of Polycomb silencing by Trx overexpression requires CBP. It was also shown that Trx and Trr are each physically associated with CBP in vivo, that Trx binds directly to the CBP KIX domain, and that the chromatin binding patterns of Trx and Trr are highly correlated with CBP and H3K4me1 genome-wide. In vitro acetylation of H3K27 by CBP is enhanced on K4me1-containing H3 substrates, and independently altering the H3K4me1 level in vivo, via the H3K4 demethylase LSD1, produces concordant changes in H3K27ac. These data indicate that the catalytic activities of Trx and CBP are physically coupled and suggest that both activities play roles in antagonizing Polycomb silencing, stimulating enhancer activity and cellular memory.
Zhang, L., Beaucher, M., Cheng, Y. and Rong, Y. S. (2014). Coordination of transposon expression with DNA replication in the targeting of telomeric retrotransposons in Drosophila. EMBO J [Epub ahead of print]. PubMed ID: 24733842
In Drosophila, a group of retrotransposons is mobilized exclusively to telomeres in a sequence-independent manner. How they target chromosome ends is not understood. This study focused on the telomeric element HeT-A and characterized the cell cycle expression and cytological distribution of its protein and RNA products. The timing of telomere replication was determined by creating a single lacO-marked telomere, and evidence is provided suggesting that transposon expression and recruitment to telomeres is linked to telomere replication. The HeT-A-encoded ORF1p protein is expressed predominantly in S phase, particularly in early S phase. Orf1p binds HeT-A transcripts and forms spherical structures at telomeres undergoing DNA replication. HeT-A sphere formation requires Verrocchio, a putative homolog of the conserved Stn1 telomeric protein. These results suggest that coupling of telomere elongation and telomere replication is a universal feature, and raise the possibility that transposon recruitment to Drosophila telomeres is mechanistically related to telomerase recruitment in other organisms. This study also supports a co-adaptive relationship between the Drosophila host and HeT-A mobile elements.
Klenov, M. S., Lavrov, S. A., Korbut, A. P., Stolyarenko, A. D., Yakushev, E. Y., Reuter, M., Pillai, R. S. and Gvozdev, V. A. (2014). Impact of nuclear Piwi elimination on chromatin state in Drosophila melanogaster ovaries. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 24782529
The Piwi-interacting RNA (piRNA)-interacting Piwi protein is involved in transcriptional silencing of transposable elements in ovaries of Drosophila melanogaster. This study characterized the genome-wide effect of nuclear Piwi elimination on the presence of the heterochromatic H3K9me3 mark and HP1a, as well as on the transcription-associated mark H3K4me2. The results demonstrate that a significant increase in the H3K4me2 level upon nuclear Piwi loss is not accompanied by the alterations in H3K9me3 and HP1a levels for several ovarian germline-expressed transposons, suggesting that in this case Piwi prevents transcription by a mechanism distinct from H3K9 methylation. It was found that the targets of Piwi-dependent chromatin repression are mainly related to the elements that display a higher level of H3K4me2 modification in the absence of silencing, i.e. most actively transcribed elements. It was also shown that Piwi-guided silencing does not significantly influence the chromatin state of dual-strand piRNA-producing clusters. In addition, host protein-coding gene expression is essentially not affected due to the nuclear Piwi elimination, but an increase in small nuclear spliceosomal RNAs abundance was noted and involvement in their post-transcriptional regulation is suggested. This work reveals new aspects of transposon silencing in Drosophila, indicating that transcription of transposons can underpin their Piwi dependent silencing, while canonical heterochromatin marks are not obligatory for their repression.
Landais, S., D'Alterio, C. and Jones, D. L. (2014). Persistent Replicative Stress Alters Polycomb Phenotypes and Tissue Homeostasis in Drosophila melanogaster. Cell Rep [Epub ahead of print]. PubMed ID: 24746823
Polycomb group (PcG) proteins establish and maintain genetic programs that regulate cell-fate decisions. Drosophila multi sex combs (mxc) was categorized as a PcG gene based on a classical Polycomb phenotype and genetic interactions; however, a mechanistic connection between Polycomb and Mxc has not been elucidated. Hypomorphic alleles of mxc are characterized by male and female sterility and ectopic sex combs. Mxc is an important regulator of histone synthesis, and it was found that increased levels of the core histone H3 in mxc mutants result in replicative stress and a persistent DNA damage response (DDR). Germline loss, ectopic sex combs and the DDR are suppressed by reducing H3 in mxc mutants. Conversely, mxc phenotypes are enhanced when the DDR is abrogated. Importantly, replicative stress induced by hydroxyurea treatment recapitulated mxc germline phenotypes. These data reveal how persistent replicative stress affects gene expression, tissue homeostasis, and maintenance of cellular identity in vivo.
Wednesday, May 7th
Masuda-Nakagawa, L. M., Ito, K., Awasaki, T. and O'Kane, C. J. (2014). A single GABAergic neuron mediates feedback of odor-evoked signals in the mushroom body of larval Drosophila. Front Neural Circuits 8: 35. PubMed ID: 24782716
Inhibition has a central role in defining the selectivity of the responses of higher order neurons to sensory stimuli. However, the circuit mechanisms of regulation of these responses by inhibitory neurons are still unclear. In Drosophila, the mushroom bodies (MBs) are necessary for olfactory memory, and by implication for the selectivity of learned responses to specific odors. To understand the circuitry of inhibition in the calyx (the input dendritic region) of the MBs and its relationship with MB excitatory activity, the simple anatomy of the Drosophila larval olfactory system was used to identify any inhibitory inputs that could contribute to the selectivity of MB odor responses. A single neuron was found to account for all detectable GABA innervation in the calyx of the MBs, and this neuron has pre-synaptic terminals in the calyx and post-synaptic branches in the MB lobes (output axonal area). This neuron was called the larval anterior paired lateral (APL) neuron, because of its similarity to the previously described adult APL neuron. GFP reconstitution across synaptic partners (GRASP) suggests that the larval APL makes extensive contacts with the MB intrinsic neurons, Kenyon Cells (KCs), but few contacts with incoming projection neurons (PNs). Using calcium imaging of neuronal activity in live larvae, this study shows that the larval APL responds to odors in a manner that requires output from KCs. These data suggest that the larval APL is the sole GABAergic neuron that innervates the MB input region and carries inhibitory feedback from the MB output region, consistent with a role in modulating the olfactory selectivity of MB neurons.
Shinomiya, K., Karuppudurai, T., Lin, T. Y., Lu, Z., Lee, C. H. and Meinertzhagen, I. A. (2014). Candidate Neural Substrates for Off-Edge Motion Detection in Drosophila. Curr Biol. PubMed ID: 24768048
In the visual motion pathways contained within the fly's optic lobe, two cell types-T4 and T5-are the first known relay neurons to signal small-field direction-selective motion responses. These cells then feed into large tangential cells that signal wide-field motion. Recent studies have identified two types of columnar neurons in the second neuropil, or medulla, that relay input to T4 from L1, the ON-channel neuron in the first neuropil, or lamina, thus providing a candidate substrate for the elementary motion detector (EMD). Interneurons relaying the OFF channel from L1's partner, L2, to T5 are so far not known, however. This study report that multiple types of transmedulla (Tm) neurons provide unexpectedly complex inputs to T5 at their terminals in the third neuropil, or lobula. From the L2 pathway, single-column input comes from Tm1 and Tm2 and multiple-column input from Tm4 cells.
Additional input to T5 comes from Tm9, the medulla target of a third lamina interneuron, L3, providing a candidate substrate for L3's combinatorial action with L2. Most numerous, Tm2 and Tm9's input synapses are spatially segregated on T5's dendritic arbor, providing candidate anatomical substrates for the two arms of a T5 EMD circuit; Tm1 and Tm2 provide a second. Transcript profiling indicates that T5 expresses both nicotinic and muscarinic cholinoceptors, qualifying T5 to receive cholinergic inputs from Tm9 and Tm2, which both express choline acetyltransferase (ChAT). It is hypothesized that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors.
Cavanaugh, D. J., Geratowski, J. D., Wooltorton, J. R., Spaethling, J. M., Hector, C. E., Zheng, X., Johnson, E. C., Eberwine, J. H. and Sehgal, A. (2014). Identification of a circadian output circuit for rest:activity rhythms in Drosophila. Cell 157: 689-701. PubMed ID: 24766812
Though much is known about the cellular and molecular components of the circadian clock, output pathways that couple clock cells to overt behaviors have not been identified. A screen was conducted for circadian-relevant neurons in the Drosophila brain, and this study reports that cells of the pars intercerebralis (PI), a functional homolog of the mammalian hypothalamus, comprise an important component of the circadian output pathway for rest:activity rhythms. GFP reconstitution across synaptic partners (GRASP) analysis demonstrates that PI cells are connected to the clock through a polysynaptic circuit extending from pacemaker cells to PI neurons. Molecular profiling of relevant PI cells identified the corticotropin-releasing factor (CRF) homolog, DH44, as a circadian output molecule that is specifically expressed by PI neurons and is required for normal rest:activity rhythms. Notably, selective activation or ablation of just six DH44+ PI cells causes arrhythmicity. These findings delineate a circuit through which clock cells can modulate locomotor rhythms.
Ohashi, S., Morimoto, T., Suzuki, Y., Miyakawa, H. and Aonishi, T. (2014). A novel behavioral strategy, continuous biased running, during chemotaxis in Drosophila larvae. Neurosci Lett 570C: 10-15. PubMed ID: 24747684
Animals collect and integrate information from their environment, and select an appropriate strategy to elicit a behavioral response. This study investigated the behavioral strategy employed by Drosophila larvae during chemotaxis toward a food source functioning as an attractive odor source. In larvae, sharp turns have been identified as the main strategy during locomotion to odorant sources, but the existence of runs orienting toward the direction of higher odor concentrations has not been described. This study shows the existence of such a successive orientation toward an odor source, which has been termed biased running. This behavioral analysis, which examines the relationship between larval rotational velocities and larval positions relative to an attractive odor source, brings out this newly found behavioral strategy. Additionally, theoretically estimated concentration gradients of chemoattractants between left and right olfactory organs were statistically correlated with rotational velocities during biased running. Finally, computer simulations demonstrated that biased running enhances navigation accuracy. Taken together, biased running is an effective behavioral strategy during chemotaxis, and this notion may provide a new insight on how animals can efficiently approach the odor source.
Tuesday, May 6th
Janssens, D. H., Komori, H., Grbac, D., Chen, K., Koe, C. T., Wang, H. and Lee, C. Y. (2014). Earmuff restricts progenitor cell potential by attenuating the competence to respond to self-renewal factors. Development 141: 1036-1046. PubMed ID: 24550111
Despite expressing stem cell self-renewal factors, intermediate progenitor cells possess restricted developmental potential, which allows them to give rise exclusively to differentiated progeny rather than stem cell progeny. Failure to restrict the developmental potential can allow intermediate progenitor cells to revert into aberrant stem cells that might contribute to tumorigenesis. Insight into stable restriction of the developmental potential in intermediate progenitor cells could improve understanding of the development and growth of tumors, but the mechanisms involved remain largely unknown. Intermediate neural progenitors (INPs), generated by type II neural stem cells (neuroblasts) in fly larval brains, provide an in vivo model for investigating the mechanisms that stably restrict the developmental potential of intermediate progenitor cells. This study reports that the transcriptional repressor protein Earmuff (Erm) functions temporally after Brain tumor (Brat) and Numb to restrict the developmental potential of uncommitted (immature) INPs. Consistently, endogenous Erm is detected in immature INPs but undetectable in INPs. Erm-dependent restriction of the developmental potential in immature INPs leads to attenuated competence to respond to all known neuroblast self-renewal factors in INPs. The BAP chromatin-remodeling complex (see Osa) probably functions cooperatively with Erm to restrict the developmental potential of immature INPs. Together, these data lead to the conclusion that the Erm-BAP-dependent mechanism stably restricts the developmental potential of immature INPs by attenuating their genomic responses to stem cell self-renewal factors. It is proposed that restriction of developmental potential by the Erm-BAP-dependent mechanism functionally distinguishes intermediate progenitor cells from stem cells, ensuring the generation of differentiated cells and preventing the formation of progenitor cell-derived tumor-initiating stem cells.
Wang, S., Tan, K. L., Agosto, M. A., Xiong, B., Yamamoto, S., Sandoval, H., Jaiswal, M., Bayat, V., Zhang, K., Charng, W. L., David, G., Duraine, L., Venkatachalam, K., Wensel, T. G. and Bellen, H. J. (2014). The retromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration. PLoS Biol 12: e1001847. PubMed ID: 24781186
Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. This study shows that the retromer complex (see Vps35) is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. It is proposed that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity.
Melnattur, K. V., Pursley, R., Lin, T. Y., Ting, C. Y., Smith, P. D., Pohida, T. and Lee, C. H. (2014). Multiple Redundant Medulla Projection Neurons Mediate Color Vision in Drosophila. J Neurogenet [Epub ahead of print]. PubMed ID: 24766346
The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons in the optic lobe, this study has uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. A novel aversive operant conditioning assay was developed for intensity-independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs (light-emitting diodes). The flies' optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow-spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad-spectrum photoreceptors R1-R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. It is hypothesized that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate ultraviolet (UV) spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons.
Fropf, R., Zhang, J., Tanenhaus, A. K., Fropf, W. J., Siefkes, E. and Yin, J. C. (2014). Time of day influences memory formation and dCREB2 proteins in Drosophila. Front Syst Neurosci 8: 43. PubMed ID: 24744705
Many biological phenomena oscillate under the control of the circadian system, exhibiting peaks and troughs of activity across the day/night cycle. In most animal models, memory formation also exhibits this property, but the underlying neuronal and molecular mechanisms remain unclear. The dCREB2 transcription factor shows circadian regulated oscillations in its activity, and has been shown to be important for both circadian biology and memory formation. This study shows that the time-of-day (TOD) of behavioral training affects Drosophila memory formation. dCREB2 exhibits complex changes in protein levels across the daytime and nighttime, and these changes in protein abundance are likely to contribute to oscillations in dCREB2 activity and TOD effects on memory formation. The results demonstrate notable correlations between the TOD behavioral effects and the circadian profile of dCREB2 proteins. At ZT = 20, there is a significant depression in memory formation, an event which coincides with apparent increases in blocker-related species clearly visible on the Western blots. At ZT = 16, a significant increase was measured in performance. This time point correlates with the end of a window (ZT = 13–15) when nuclear levels of the activator are elevated. Based on these relationships, it is hypothesized that the dynamics of dCREB2 protein levels contribute to the TOD effects on memory formation.
Monday, May 5th
Feng, Y., Li, X., Ray, L., Song, H., Qu, J., Lin, S. and Lin, X. (2014). The Drosophila Tankyrase regulates Wg Signaling depending on the concentration of Daxin. Cell Signal [Epub ahead of print]. PubMed ID: 24768997
The canonical Wnt signaling pathway plays critical roles during development and homeostasis. Dysregulation of this pathway can lead to many human diseases, including cancers. A key process in this pathway consists of regulation of beta-catenin concentration through an Axin-recruited destruction complex. Previous studies have demonstrated a role for Tankyrase (TNKS), a protein with poly (ADP-ribose) polymerase, in the regulation of Axin levels in human cancers. However, the role of TNKS in development is still unclear. This study generated a Drosophila tankyrase (DTNKS) mutant and provided compelling evidence that DTNKS is involved in the degradation of Drosophila Axin (Daxin). Daxin was shown to physically interact with DTNKS, and its protein levels are elevated in the absence of DTNKS in eye discs. In S2 cells, DTNKS suppressed the levels of Daxin. Surprisingly, it was found that Daxin in turn down-regulated DTNKS protein level. In vivo study showed that DTNKS regulated Wg signaling and wing patterning at a high Daxin protein level, but not at a normal level. Taken together, these findings identified a conserved role of DTNKS in regulating Daxin levels, and thereby Wg/Wnt signaling during development.
Ribeiro, P., Holder, M., Frith, D., Snijders, A. P. and Tapon, N. (2014). Crumbs promotes expanded recognition and degradation by the SCFSlimb/beta-TrCP ubiquitin ligase. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 24778256
In epithelial tissues, growth control depends on the maintenance of proper architecture through apicobasal polarity and cell-cell contacts. The Hippo signaling pathway has been proposed to sense tissue architecture and cell density via an intimate coupling with the polarity and cell contact machineries. The apical polarity protein Crumbs (Crb) controls the activity of Yorkie (Yki)/Yes-activated protein, the progrowth target of the Hippo pathway core kinase cassette, both in flies and mammals. The apically localized Four-point-one, Ezrin, Radixin, Moesin domain protein Expanded (Ex) regulates Yki by promoting activation of the kinase cascade and by directly tethering Yki to the plasma membrane. Crb interacts with Ex and promotes its apical localization, thereby linking cell polarity with Hippo signaling. This study shows that, as well as repressing Yki by recruiting Ex to the apical membrane, Crb promotes phosphorylation-dependent ubiquitin-mediated degradation of Ex. Skp/Cullin/F-boxSlimb/beta-transducin repeats-containing protein (SCFSlimb/beta-TrCP) was identifed as the E3 ubiquitin ligase complex responsible for Ex degradation. Thus, Crb is part of a homeostatic mechanism that promotes Ex inhibition of Yki, but also limits Ex activity by inducing its degradation, allowing precise tuning of Yki function.
Claret, S., Jouette, J., Benoit, B., Legent, K. and Guichet, A. (2014). PI(4,5)P2 Produced by the PI4P5K SKTL Controls Apical Size by Tethering PAR-3 in Drosophila Epithelial Cells. Curr Biol [Epub ahead of print]. PubMed ID: 24768049
The control of apical-basal polarity in epithelial layers is a fundamental event in many processes, ranging from embryonic development to tumor formation. A key feature of polarized epithelial cells is their ability to maintain an asymmetric distribution of specific molecular complexes, including the phosphoinositides PI(4,5)P2 and PI(3,4,5)P3. The spatiotemporal regulation of these phosphoinositides is controlled by the concerted action of phosphoinositide kinases and phosphatases. Using the Drosophila follicular epithelium as a model system in vivo, this study shows that PI(4,5)P2 is crucial to maintain apical-basal polarity. PI(4,5)P2 is essentially regulated by the PI4P5 kinase Skittles (SKTL), whereas neither the phosphatase PTEN nor the PI(4,5)P3 kinase DP110 lead to loss of apical-basal polarity. By inactivating SKTL and thereby strongly reducing PI(4,5)P2 levels in a single cell of the epithelium, the disassembly was observed of adherens junctions, actin cytoskeleton reorganization, and apical constriction leading to delamination, a process similar to that observed during epithelial-mesenchymal transition. Evidence is provided that PI(4,5)P2 controls the apical targeting of PAR-3/Bazooka to the plasma membrane and that the loss of this polarized distribution is sufficient to induce a similar cell shape change. Finally, it was shown that PI(4,5)P2 is excluded from the cell apex and that PAR-3 diffuses laterally just prior to the apical constriction in a context of endogenous invagination. All together, these results indicate that the PIP5 kinase SKTL, by controlling PI(4,5)P2 polarity, regulates PAR-3 localization and thus the size of the apical domain.
Kuzhandaivel, A., Schultz, S. W., Alkhori, L. and Alenius, M. (2014). Cilia-Mediated Hedgehog Signaling in Drosophila. Cell Rep [Epub ahead of print]. PubMed ID: 24768000
Cilia mediate Hedgehog (Hh) signaling in vertebrates and Hh deregulation results in several clinical manifestations, such as obesity, cognitive disabilities, developmental malformations, and various cancers. Drosophila cells are nonciliated during development, which has led to the assumption that cilia-mediated Hh signaling is restricted to vertebrates. This study identified and characterized a cilia-mediated Hh pathway in Drosophila olfactory sensory neurons. Several fundamental key aspects of the vertebrate cilia pathway, such as ciliary localization of Smoothened and the requirement of the intraflagellar transport system, are present in Drosophila. Cos2 and Fused are required for the ciliary transport of Smoothened and cilia mediate the expression of the Hh pathway target genes. Taken together, these data demonstrate that Hh signaling in Drosophila can be mediated by two pathways and that the ciliary Hh pathway is conserved from Drosophila to vertebrates.
Sunday, May 4th
Guo, T., Lu, Y., Li, P., Yin, M. X., Lv, D., Zhang, W., Wang, H., Zhou, Z., Ji, H., Zhao, Y. and Zhang, L. (2013). A novel partner of Scalloped regulates Hippo signaling via antagonizing Scalloped-Yorkie activity. Cell Res 23: 1201-1214. PubMed ID: 23999857
The Hippo (Hpo) pathway controls tissue growth and organ size by regulating the activity of transcriptional co-activator Yorkie (Yki), which associates with transcription factor Scalloped (Sd) in the nucleus to promote downstream target gene expression. This study identified a novel protein Sd-Binding-Protein (SdBP)/Tgi, which directly competes with Yki for binding to Sd through its TDU domains and inhibits the Sd-Yki transcriptional activity. SdBP was found to retain Yki in the nucleus through the association with Yki WW domains via its PPXY motifs. Collectively, SdBP was identified as a novel component of the Hpo pathway, negatively regulating the transcriptional activity of Sd-Yki to restrict tissue growth.
Wei, Y. and Lilly, M. A. (2014). The TORC1 inhibitors Nprl2 and Nprl3 mediate an adaptive response to amino-acid starvation in Drosophila. Cell Death Differ [Epub ahead of print]. PubMed ID: 24786828
Target of rapamycin complex 1 (TORC1; see Tor) is a master regulator of metabolism in eukaryotes that integrates information from multiple upstream signaling pathways. In yeast, the Nitrogen permease regulators 2 and 3 (Npr2 and Npr3) mediate an essential response to amino-acid limitation upstream of TORC1. In mammals, the Npr2 ortholog, Nprl2, is a putative tumor suppressor gene that inhibits cell growth and enhances sensitivity to numerous anticancer drugs including cisplatin. However, the precise role of Nprl2 and Nprl3 in the regulation of metabolism in metazoans remains poorly defined. This study demonstrates that the central importance of Nprl2 and Nprl3 in the response to amino-acid starvation has been conserved from single celled to multicellular animals. In Drosophila Nprl2/CG9104 and Nprl3/CG31183 physically interact and are targeted to lysosomes and autolysosomes. Using oogenesis as a model system, this study showed that Nprl2 and Nprl3 inhibit TORC1 signaling in the female germline in response to amino-acid starvation. Moreover, the inhibition TORC1 by Nprl2/3 is critical to the preservation of female fertility during times of protein scarcity. In young egg chambers the failure to downregulate TORC1 in response to amino-acid limitation triggers apoptosis. Thus, these data suggest the presence of a metabolic checkpoint that initiates a cell death program when TORC1 activity remains inappropriately high during periods of amino-acid and/or nutrient scarcity in oogenesis. Finally, it was demonstrated that Nprl2/3 work in concert with the TORC1 inhibitors Tsc1/2 to fine tune TORC1 activity during oogenesis and that Tsc1 is a critical downstream effector of Akt1 in the female germline.
Tchankouo-Nguetcheu, S., Udinotti, M., Durand, M., Meng, T. C., Taouis, M. and Rabinow, L. (2014). Negative regulation of MAP kinase signaling in Drosophila by Ptp61F/PTP1B. Mol Genet Genomics [Epub ahead of print]. PubMed ID: 24752400
PTP1B is an important negative regulator of insulin and other signaling pathways in mammals. However, the role of PTP1B in the regulation of RAS-MAPK signaling remains open to deliberation, due to conflicting evidence from different experimental systems. The Drosophila orthologue of mammalian PTP1B, PTP61F, has until recently remained largely uncharacterized. To establish the potential role of PTP61F in the regulation of signaling pathways in Drosophila and particularly to help resolve its fundamental function in RAS-MAPK signaling, a new allele of Ptp61F, as well as both RNA interference and overexpression alleles were generated. The results validate recent data showing that the activity of insulin and Abl kinase signaling is increased in Ptp61F mutants and RNA interference lines. Importantly, this study established negative regulation of the RAS/MAPK pathway by Ptp61F activity in whole animals. Of particular interest, the results document the modulation of hyperactive MAP kinase activity by Ptp61F alleles, showing that the phosphatase intervenes to directly or indirectly regulate MAP kinase itself.
Yanai, H., Yoshioka, Y., Yoshida, H., Nakao, Y., Plessis, A. and Yamaguchi, M. (2014). Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway. Oncogenesis 3: e98. PubMed ID: 24752236
Drosophila Myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. This study found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, evidence is provided that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. It was shown that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis.
Saturday, May 3rd
Yang, L., Li, R., Kaneko, T., Takle, K., Morikawa, R. K., Essex, L., Wang, X., Zhou, J., Emoto, K., Xiang, Y. and Ye, B. (2014). Trim9 Regulates Activity-Dependent Fine-Scale Topography in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 24746793
Topographic projection of afferent terminals into 2D maps in the CNS is a general strategy used by the nervous system to encode the locations of sensory stimuli. In vertebrates, it is known that although guidance cues are critical for establishing a coarse topographic map, neural activity directs fine-scale topography between adjacent afferent terminals. However, the molecular mechanism underlying activity-dependent regulation of fine-scale topography is poorly understood. Molecular analysis of the spatial relationship between adjacent afferent terminals requires reliable localization of the presynaptic terminals of single neurons as well as genetic manipulations with single-cell resolution in vivo. Although both requirements can potentially be met in Drosophila melanogaster, no activity-dependent topographic system has been identified in flies. This study reports a topographic system that is shaped by neuronal activity in Drosophila. With this system, it was found that topographic separation of the presynaptic terminals of adjacent nociceptive neurons requires different levels of Trim9, an evolutionarily conserved, ring finger domain-containing, multi-domain signaling molecule. Neural activity regulates Trim9 protein levels to direct fine-scale topography of sensory afferents. This study offers both a novel mechanism by which neural activity directs fine-scale topography of axon terminals and a new system to study this process at single-neuron resolution.
Pecot, M. Y., Chen, Y., Akin, O., Chen, Z., Tsui, C. Y. and Zipursky, S. L. (2014). Sequential axon-derived signals couple target survival and layer specificity in the Drosophila visual system. Neuron 82: 320-333. PubMed ID: 24742459
Neural circuit formation relies on interactions between axons and cells within the target field. While it is well established that target-derived signals act on axons to regulate circuit assembly, the extent to which axon-derived signals control circuit formation is not known. In the Drosophila visual system, anterograde signals numerically match R1-R6 photoreceptors with their targets by controlling target proliferation and neuronal differentiation. This study demonstrates that additional axon-derived signals selectively couple target survival with layer specificity. Jelly belly (Jeb) produced by R1-R6 axons was shown to interact with its receptor, Anaplastic lymphoma kinase (Alk), on budding dendrites to control survival of L3 neurons, one of three postsynaptic targets. L3 axons then produce Netrin, which regulates the layer-specific targeting of another neuron within the same circuit. It is proposed that a cascade of axon-derived signals, regulating diverse cellular processes, provides a strategy for coordinating circuit assembly across different regions of the nervous system.
Honda, T., Lee, C. Y., Yoshida-Kasikawa, M., Honjo, K. and Furukubo-Tokunaga, K. (2014). Induction of associative olfactory memory by targeted activation of single olfactory neurons in Drosophila larvae. Sci Rep 4: 4798. PubMed ID: 24762789
It has been postulated that associative memory is formed by at least two sets of external stimuli, CS and US, that are transmitted to the memory centers by distinctive conversing pathways. However, whether associative memory can be induced by the activation of only the olfactory CS and a biogenic amine-mediated US pathways remains to be elucidated. In this study, the reward signals were substituted with dTrpA1-mediated thermogenetic activation of octopaminergic neurons and the odor signals by ChR2-mediated optical activation of a specific class of olfactory neurons. Targeted activation of the olfactory receptor and the octopaminergic neurons was shown to be indeed sufficient for the formation of associative olfactory memory in the larval brain. It was also shown that targeted stimulation of only a single type of olfactory receptor neuron is sufficient to induce olfactory memory that is indistinguishable from natural memory induced by the activation of multiple olfactory receptor neurons.
Selcho, M., Pauls, D., Huser, A., Stocker, R. F. and Thum, A. S. (2014). Characterization of the octopaminergic and tyraminergic neurons in the central brain of Drosophila larvae. J Comp Neurol [Epub ahead of print]. PubMed ID: 24752702
Drosophila larvae are able to evaluate sensory information based on prior experience, similar to adult flies, other insect species and vertebrates. Larvae and adult flies can be taught to associate odor stimuli with sugar reward and prior work has implicated both the octopaminergic and dopaminergic modulatory systems in reinforcement signaling. This study used genetics to analyze the anatomy, up to the single-cell level, of the octopaminergic/tyraminergic system in the larval brain and suboesophageal ganglion. Genetic ablation of subsets of these neurons allowed determination of their necessity for appetitive olfactory learning. These experiments reveal that a small subset of about 39 largely morphologically distinguishable octopaminergic/tyraminergic neurons is involved in signaling reward in the Drosophila larval brain. In addition to prior work on larval locomotion, these data functionally separate the octopaminergic/tyraminergic system into two sets of about 40 neurons. Those situated in the thoracic/abdominal ganglion are involved in larva locomotion, whereas the others in the suboesophageal ganglion and brain hemispheres mediate reward signaling.
Saturday, May 2nd
Hauling, T., Krautz, R., Markus, R., Volkenhoff, A., Kucerova, L. and Theopold, U. (2014). A Drosophila immune response against Ras-induced overgrowth. Biol Open [Epub ahead of print]. PubMed ID: 24659248
This study set out to characterize the innate immune response against the early stage of tumor development. For this, animal models where genetic changes in specific cells and tissues can be performed in a controlled way have become increasingly important, including the fruitfly Drosophila melanogaster. Many tumor mutants in Drosophila affect the germline and, as a consequence, also the immune system itself, making it difficult to ascribe their phenotype to a specific tissue. Only during the past decade, mutations have been induced systematically in somatic cells to study the control of tumorous growth by neighboring cells and by immune cells. This study shows that upon ectopic expression of a dominant-active form of the Ras oncogene (RasV12), both imaginal discs and salivary glands are affected. Particularly, the glands increase in size, express metalloproteinases and display apoptotic markers. This leads to a strong cellular response, which has many hallmarks of the granuloma-like encapsulation reaction, usually mounted by the insect against larger foreign objects. RNA sequencing of the fat body reveals a characteristic humoral immune response. In addition, genes were identified that are specifically induced upon expression of RasV12. As a proof-of-principle, it was shown that one of the induced genes (the scavenger receptor santa-maria), modulates damage to the salivary glands. The list of genes identified in this study provides a rich source for further functional characterization. It is hoped that this work will lead to a better understanding of the earliest stage of innate immune responses against tumors with implications for mammalian immunity.
Combe, B. E., Defaye, A., Bozonnet, N., Puthier, D., Royet, J. and Leulier, F. (2014). Drosophila Microbiota Modulates Host Metabolic Gene Expression via IMD/NF-kappaB Signaling. PLoS One 9: e94729. PubMed ID: 24733183
Most metazoans engage in mutualistic interactions with their intestinal microbiota. Despite recent progress the molecular mechanisms through which microbiota exerts its beneficial influences on host physiology are still largely uncharacterized. This study uses axenic Drosophila melanogaster adults associated with a standardized microbiota composed of a defined set of commensal bacterial strains to study the impact of microbiota association on its host transcriptome. The results demonstrate that Drosophila microbiota has a marked impact on the midgut transcriptome and promotes the expression of genes involved in host digestive functions and primary metabolism. The IMD/Relish signaling pathway was identified as a central regulator of this microbiota-mediated transcriptional response, and a marked transcriptional trade-off was revealed between the midgut response to its beneficial microbiota and to bacterial pathogens. Taken together these results indicate that microbiota association potentiates host nutrition and host metabolic state, two key physiological parameters influencing host fitness. This work paves the way to subsequent mechanistic studies to reveal how these microbiota-dependent transcriptional signatures translate into host physiological benefits.
Obata, F., Kuranaga, E., Tomioka, K., Ming, M., Takeishi, A., Chen, C. H., Soga, T. and Miura, M. (2014). Necrosis-Driven Systemic Immune Response Alters SAM Metabolism through the FOXO-GNMT Axis. Cell Rep [Epub ahead of print]. PubMed ID: 24746817
Sterile inflammation triggered by endogenous factors is thought to contribute to the pathogenesis of acute and chronic inflammatory diseases. This study demonstrates that apoptosis-deficient mutants (hypomorphic Apaf-1 mutants) spontaneously develop a necrosis-driven systemic immune response in Drosophila and provides an in vivo model for studying the organismal response to sterile inflammation. Metabolomic analysis of hemolymph from apoptosis-deficient mutants revealed increased sarcosine and reduced S-adenosyl-methionine (SAM) levels due to glycine N-methyltransferase (Gnmt) upregulation. Gnmt was elevated in response to Toll activation induced by the local necrosis of wing epidermal cells. Necrosis-driven inflammatory conditions induced dFoxO hyperactivation, leading to an energy-wasting phenotype. Gnmt was cell-autonomously upregulated by dFoxO in the fat body as a possible rheostat for controlling energy loss, which functioned during fasting as well as inflammatory conditions. It is proposed that the dFoxO-Gnmt axis is essential for the maintenance of organismal SAM metabolism and energy homeostasis.
Parthier, C., Stelter, M., Ursel, C., Fandrich, U., Lilie, H., Breithaupt, C. and Stubbs, M. T. (2014). Structure of the Toll-Spatzle complex, a molecular hub in Drosophila development and innate immunity. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 24733933
Drosophila Toll receptors are involved in embryonic development and the immune response of adult flies. In both processes, the only known Toll receptor ligand is the human nerve growth factor-like cystine knot protein Spatzle. This study presents the crystal structure of a 1:1 (nonsignaling) complex of the full-length Toll receptor ectodomain (ECD) with the Spatzle cystine knot domain dimer. The ECD is divided into two leucine-rich repeat (LRR) domains, each of which is capped by cysteine-rich domains. Spatzle binds to the concave surface of the membrane-distal LRR domain, in contrast to the flanking ligand interactions observed for mammalian Toll-like receptors, with asymmetric contributions from each Spatzle protomer. The structure allows rationalization of existing genetic and biochemical data and provides a framework for targeting the immune systems of insects of economic importance, as well as a variety of invertebrate disease vectors.
Thursday, April 1st
Lawton, K. J., Wassmer, T. L. and Deitcher, D. L. (2014). Conserved role of Drosophila melanogaster FoxP in motor coordination and courtship song. Behav Brain Res [Epub ahead of print]. PubMed ID: 24747661
FoxP2 is a highly conserved vertebrate transcription factor known for its importance in human speech and language production. Disruption of FoxP2 in several vertebrate models indicates a conserved functional role for this gene in both sound production and motor coordination. Although FoxP2 is known to be strongly expressed in brain regions important for motor coordination, little is known about FoxP2's role in the nervous system. The recent discovery of the well-conserved Drosophila melanogaster homolog, FoxP, provides an opportunity to study the role of this crucial gene in an invertebrate model. It was hypothesized that, like FoxP2, Drosophila FoxP is important for behaviors requiring fine motor coordination. This study used targeted RNA interference to reduce expression of FoxP, and the effects were assayed on a variety of adult behaviors. Male flies with reduced FoxP expression exhibit decreased levels of courtship behavior, altered pulse-song structure, and sex-specific motor impairments in walking and flight. Acute disruption of synaptic activity in FoxP expressing neurons using a temperature-sensitive shibire allele dramatically impaired motor coordination. Utilizing a GFP reporter to visualize FoxP in the fly brain reveals expression in relatively few neurons in distributed clusters within the larval and adult CNS, including distinct labeling of the adult protocerebral bridge - a section of the insect central complex known to be important for motor coordination and thought to be homologous to areas of the vertebrate basal ganglia. These results establish the necessity of this gene in motor coordination in an invertebrate model and suggest a functional homology with vertebrate FoxP2.
Kayser, M. S., Yue, Z. and Sehgal, A. (2014). A critical period of sleep for development of courtship circuitry and behavior in Drosophila. Science 344: 269-274. PubMed ID: 24744368
Most animals sleep more early in life than in adulthood, but the function of early sleep is not known. Using Drosophila, this study found that increased sleep in young flies was associated with an elevated arousal threshold and resistance to sleep deprivation. Excess sleep results from decreased inhibition of a sleep-promoting region by a specific dopaminergic circuit. Experimental hyperactivation of this circuit in young flies results in sleep loss and lasting deficits in adult courtship behaviors. These deficits are accompanied by impaired development of a single olfactory glomerulus, VA1v, which normally displays extensive sleep-dependent growth after eclosion. These results demonstrate that sleep promotes normal brain development that gives rise to an adult behavior critical for species propagation and suggest that rapidly growing regions of the brain are most susceptible to sleep perturbations early in life.
Lamba, P., Bilodeau-Wentworth, D., Emery, P. and Zhang, Y. (2014). Morning and Evening Oscillators Cooperate to Reset Circadian Behavior in Response to Light Input. Cell Rep. PubMed ID: 24746814
Light is a crucial input for circadian clocks. In Drosophila, short light exposure can robustly shift the phase of circadian behavior. The model for this resetting posits that circadian photoreception is cell autonomous: Cryptochrome senses light, binds to Timeless (Tim), and promotes its degradation, which is mediated by Jetlag (Jet). However, it was recently proposed that interactions between circadian neurons are also required for phase resetting. This study identified two groups of neurons critical for circadian photoreception: the morning (M) and the evening (E) oscillators. These neurons work synergistically to reset rhythmic behavior. Jet promotes acute Tim degradation cell autonomously in M and E oscillators but also nonautonomously in E oscillators when expressed in M oscillators. Thus, upon light exposure, the M oscillators communicate with the E oscillators. Because the M oscillators drive circadian behavior, they must also receive inputs from the E oscillators. Hence, although photic Tim degradation is largely cell autonomous, neural cooperation between M and E oscillators is critical for circadian behavioral photoresponses.
Sun, F., Wang, Y., Zhou, Y., Van Swinderen, B., Gong, Z. and Liu, L. (2014). Identification of neurons responsible for feeding behavior in the Drosophila brain. Sci China Life Sci [Epub ahead of print]. PubMed ID: 24744088
Drosophila melanogaster feeds mainly on rotten fruits, which contain many kinds of sugar. Thus, the sense of sweet taste has evolved to serve as a dominant regulator and driver of feeding behavior. Although several sugar receptors have been described, it remains poorly understood how the sensory input is transformed into an appetitive behavior. This study used a neural silencing approach to screen brain circuits, and identified neurons labeled by three Gal4 lines that modulate Drosophila feeding behavior. These three Gal4 lines labeled neurons mainly in the suboesophageal ganglia (SOG), which is considered to be the fly's primary taste center. When the activity of these neurons was blocked, flies decreased their sugar consumption significantly. In contrast, activation of these neurons resulted in enhanced feeding behavior and increased food consumption not only towards sugar, but to an array of food sources. Moreover, upon neuronal activation, the flies demonstrated feeding behavior even in the absence of food, which suggests that neuronal activation can replace food as a stimulus for feeding behavior. These findings indicate that these Gal4-labeled neurons, which function downstream of sensory neurons and regulate feeding behavior towards different food sources is necessary in Drosophila feeding control.
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