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


Monday, November 30th, 2015

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Swanson, C. I., Meserve, J. H., McCarter, P. C., Thieme, A., Mathew, T., Elston, T. C. and Duronio, R. J. (2015). Expression of an S phase-stabilized version of the CDK inhibitor Dacapo can alter endoreplication. Development [Epub ahead of print]. PubMed ID: 26493402
In developing organisms, divergence from the canonical cell division cycle is often necessary to ensure the proper growth, differentiation, and physiological function of a variety of tissues. An important example is endoreplication, in which endocycling cells alternate between G and S phase without intervening mitosis or cytokinesis, resulting in polyploidy. Although significantly different from the canonical cell cycle, endocycles use regulatory pathways that also function in diploid cells, particularly those involved in S phase entry and progression. A key S phase regulator is the Cyclin E/Cdk2 kinase, which must alternate between periods of high (S phase) and low (G phase) activity in order for endocycling cells to achieve repeated rounds of S phase and polyploidy. The mechanisms that drive these oscillations of Cyclin E/Cdk2 activity are not fully understood. This study shows that the Drosophila Cyclin E/Cdk2 inhibitor Dacapo is targeted for destruction during S phase via a PIP degron, contributing to oscillations of Dap protein accumulation during both mitotic cycles and endocycles. Expression of a PIP degron mutant Dap attenuates endocycle progression but does not obviously affect proliferating diploid cells. A mathematical model of the endocycle predicts that the rate of destruction of Dap during S phase modulates the endocycle by regulating the length of G phase. It is proposed from this model and the in vivo data that endo S phase-coupled destruction of Dap reduces the threshold of Cyclin E/Cdk2 activity necessary to trigger the subsequent G-S transition, thereby influencing endocycle oscillation frequency and the extent of polyploidy.

Giansanti, M. G., et al. (2015). Exocyst-dependent membrane addition is required for anaphase cell elongation and cytokinesis in Drosophila. PLoS Genet 11: e1005632. PubMed ID: 26528720
Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. This study investigated the functional requirements for exocyst complex function during cell division in vivo, and demonstrates a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. This study used high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, it was observed that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, it was shown that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. These results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.

Wang, P. Y.,Lin, W. C., Tsai, Y. C., Cheng, M. L., Lin, Y. H., Tseng, S. H., Chakraborty, A. and Pai, L. M. (2015). Regulation of CTP synthase filament formation during DNA endoreplication in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 26482795
CTP synthase (CTPsyn) plays an essential role in DNA, RNA, and lipid synthesis. A polymeric CTPsyn structure dynamically regulates its enzymatic activity. This study found that reversible ubiquitination regulates the dynamic assembly of the filamentous structures of Drosophila CTPsyn. It was further determined that the proto-oncogene Cbl, an E3 ubiquitin ligase, controls CTPsyn filament formation in endocycles. While the E3 ligase activity of Cbl is required for CTPsyn filament formation, Cbl does not affect the protein levels of CTPsyn. It remains unclear whether the regulation of CTPsyn filaments by Cbl is through direct ubiquitination of CTPsyn. In the absence of Cbl or with knockdown of CTPsyn, the progression of the endocycle-associated S phase was impaired. Furthermore, overexpression of wild-type, but not enzymatically inactive CTPsyn, rescued the endocycle defect in Cbl mutant cells. Together, these results suggest that Cbl influences the nucleotide pool balance and controls CTPsyn filament formation in endocycles. This study links Cbl-mediated ubiquitination to the polymerization of a metabolic enzyme, and reveals a role for Cbl in endocycles during Drosophila development.

Conduit, P. T., Wainman, A., Novak, Z. A., Weil, T. T. and Raff, J. W. (2015). Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP. Elife 4 [Epub ahead of print]. PubMed ID: 26530814
Centrosomes have many important functions and comprise a 'mother' and 'daughter' centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic 'S-CAP' complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) it was shown that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. This study shows , however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes. Importantly, Sas-4 may have a more indirect role in mitotic PCM assembly in fly embryos as centriolar Sas-4 (as opposed to cytoplasmic Sas-4) is required to efficiently recruit Asl molecules around maturing mother centrioles, and Asl has an important role in recruiting Spd-2 and Cnn around mother centrioles

Sunday, November 29th

Parchure, A., Vyas, N., Ferguson, C., Parton, R. G. and Mayor, S. (2015). Oligomerization and endocytosis of Hedgehog is necessary for its efficient exovesicular secretion. Mol Biol Cell. PubMed ID: 26490120
Hedgehog (Hh) is a secreted morphogen, involved in both short and long range signaling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. Previous work has shown that the long range signaling of Hh requires its oligomerization. This study shows that Hh is secreted in the form of exovesicles. These are derived by the endocytic delivery of cell surface Hh to multi vesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT: see Hrs)-dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signaling in Drosophila wing imaginal discs. Importantly oligomerization-defective Hh is inefficiently incorporated into exovesicles due to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long range signaling.

Gradilla, A. C., et al. (2014). Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion. Nat Commun 5: 5649. PubMed ID: 25472772
The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. This study shows that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. It is proposed that Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen.

Kelley, C. F., Becalska, A. N., Berciu, C., Nicastro, D. and Rodal, A. A. (2015). Assembly of actin filaments and microtubules in Nwk F-BAR-induced membrane deformations. Commun Integr Biol 8: e1000703. PubMed ID: 26478768
F-BAR domains form crescent-shaped dimers that bind to and deform lipid bilayers, and play a role in many cellular processes requiring membrane remodeling, including endocytosis and cell morphogenesis. Nervous Wreck (Nwk) encodes an F-BAR/SH3 protein that regulates synapse growth in Drosophila. Unlike conventional F-BAR proteins that assemble tip-to-tip into filaments and helical arrays around membrane tubules, the Nwk F-BAR domain instead assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. In cells, this membrane deforming activity generates small buds, which can lengthen into extensive protrusions upon actin cytoskeleton polymerization. This study shows that Nwk-induced cellular protrusions contain dynamic microtubules, distinguishing them from conventional filopodia, and further do not depend on actin filaments or microtubules for their maintenance. These results indicate new ways in which close cooperation between the membrane remodeling and cytoskeletal machinery underlies large-scale changes in cellular morphology.

Lincoln, B. L., Alabsi, S. H., Frendo, N., Freund, R. and Keller, L. C. (2015). Drosophila neuronal injury follows a temporal sequence of cellular events leading to degeneration at the neuromuscular junction. J Exp Neurosci 9: 1-9. PubMed ID: 26512206
There is a critical need to improve understanding of the molecular and cellular mechanisms that drive neurodegeneration. At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components. This study used an in vivo motor neuron injury assay to acutely induce neurodegeneration in order to follow the temporal order of events that occur following injury in Drosophila. Sites of injury can be rapidly identified based on structural defects to the neuronal cytoskeleton that result in disrupted axonal transport. Additionally, the neuromuscular junction accumulates ubiquitinated proteins prior to the neurodegenerative events, occurring at 24 hours post injury. These data provide insights into the early molecular events that occur during axonal and neuromuscular degeneration in a genetically tractable model organism. Importantly, the mechanisms that mediate neurodegeneration in flies are conserved in humans. Thus, these studies will facilitate the identification of biomedically relevant targets for future treatments.

Saturday, November 28th

Hermant, C., Boivin, A., Teysset, L., Delmarre, V., Asif-Laidin, A., Beek, M. V., Antoniewski, C. and Ronsseray, S. (2015). Paramutation in Drosophila requires both nuclear and cytoplasmic actors of the piRNA pathway and induces cis-spreading of piRNA production. Genetics [Epub ahead of print]. PubMed ID: 26482790
Transposable element (TE) activity is repressed in the germline in animals by PIWI-Interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. In Drosophila, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. This paper reports that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21nt RNAs which are produced by the paramutated cluster in addition to 23-28nt piRNAs are not necessary for paramutation to occur. Production of these 21nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus which produces piRNAs along the whole length of the transgenes. Finally, it was observed that maternally-inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between non-fully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations.

Lin, C. J., Smibert, P., Zhao, X., Hu, J. F., Ramroop, J., Kellner, S. M., Benton, M. A., Govind, S., Dedon, P. C., Sternglanz, R. and Lai, E. C. (2015). An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis. RNA 21: 2103-2118. PubMed ID: 26516084
N(6)-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon-codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. This study reports the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however,substantial t6A-modified tRNAs is still detected even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, prominent kae1 melanotic masses were characterized and shown to be associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. These studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease.

Pek, J. W., Osman, I., Tay, M. L. and Zheng, R. T. (2015). Stable intronic sequence RNAs have possible regulatory roles in Drosophila melanogaster. J Cell Biol 211: 243-251. PubMed ID: 26504165
Stable intronic sequence RNAs (sisRNAs) have been found in Xenopus tropicalis, human cell lines, and Epstein-Barr virus; however, the biological significance of sisRNAs remains poorly understood. This study identifies sisRNAs in Drosophila melanogaster by deep sequencing, reverse transcription polymerase chain reaction, and Northern blotting. A sisRNA (sisR-1) was identified from the regena (rga) locus, it was shown to be processed from the precursor messenger RNA (pre-mRNA). A cis-natural antisense transcript (ASTR) was identified from the rga locus, which is highly expressed in early embryos. During embryogenesis, ASTR promotes robust rga pre-mRNA expression. Interestingly, sisR-1 represses ASTR, with consequential effects on rga pre-mRNA expression. The results suggest a model in which sisR-1 modulates its host gene expression by repressing ASTR during embryogenesis. It is proposed that sisR-1 belongs to a class of sisRNAs with probable regulatory activities in Drosophila.

Sun, X., Yang, H., Sturgill, D., Oliver, B., Rabinow, L. and Samson, M. L. (2015). Sxl-dependent, tra/tra2-independent alternative splicing of the Drosophila melanogaster X-Linked gene found in neurons.G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26511498
Somatic sexual determination and behavior in Drosophila melanogaster are under the control of a genetic cascade initiated by Sex lethal (Sxl). In the female soma, SXL RNA binding-protein regulates the splicing of transformer (tra) transcripts into a female-specific form. The RNA binding protein TRA and its cofactor TRA2 function in concert in females, whereas SXL, TRA and TRA2 are thought not to function in males. To better understand sex-specific regulation of gene expression, this study analyzed male and female head transcriptome datasets for expression levels and splicing, quantifying sex-biased gene expression via RNA-Seq and qPCR. The data uncouples the effects of Sxl and tra/tra2 in females in the sex-biased alternative splicing of head transcripts from the X-linked locus found in neurons (fne), encoding a pan-neuronal RNA-binding protein of the ELAV family. FNE protein levels are down regulated by Sxl in female heads, also independently of tra/tra2. It is argued that this regulation may have important sexually dimorphic consequences for the regulation of nervous system development or function.

Friday, November 27th

Dolezal, D., Liu, Z., Zhou, Q. and Pignoni, F. (2015). Fly LMBR1/LIMR-type protein Lilipod promotes germ-line stem cell self-renewal by enhancing BMP signaling. Proc Natl Acad Sci U S A 112: 13928-13933. PubMed ID: 26512105
Limb development membrane protein-1 (LMBR1)/lipocalin-interacting membrane receptor (LIMR)-type proteins are putative nine-transmembrane receptors that are evolutionarily conserved across metazoans. However, their biological function is unknown. This study shows that the fly family member Lilipod (Lili; CG5807) is required for germ-line stem cell (GSC) self-renewal in the Drosophila ovary where it enhances bone morphogenetic protein (BMP) signaling. lili mutant GSCs are lost through differentiation, and display reduced levels of the Dpp transducer pMad and precocious activation of the master differentiation factor bam. Conversely, overexpressed Lili induces supernumerary pMad-positive bamP-GFP-negative GSCs. Interestingly, differentiation of lili mutant GSCs is bam-dependent; however, its effect on pMad is not. Thus, although it promotes stem cell self-renewal by repressing a bam-dependent process, Lilipod enhances transduction of the Dpp signal independently of its suppression of differentiation. In addition, because Lili is still required by a ligand-independent BMP receptor, its function likely occurs between receptor activation and pMad phosphorylation within the signaling cascade. This first in vivo characterization of a LMBR1/LIMR-type protein in a genetic model reveals an important role in modulating BMP signaling during the asymmetric division of an adult stem cell population and in other BMP signaling contexts.

Xiang, W., Zhang, D. and Montell, D. J. (2015). Tousled-like kinase regulates cytokine-mediated communication between cooperating cell types during collective border cell migration.Mol Biol Cell [Epub ahead of print]. PubMed ID: 26510500
Collective cell migration is emerging as a major contributor to normal development and disease. Collective movement of border cells in the Drosophila ovary requires cooperation between two distinct cell types: 4-6 migratory cells surrounding two immotile cells called polar cells. Polar cells secrete a cytokine, Unpaired (Upd), which activates JAK/STAT signaling in neighboring cells, stimulating their motility. Without Upd, migration fails, causing sterility. Ectopic Upd expression is sufficient to stimulate motility in otherwise immobile cells. Thus regulation of Upd is key. This study reports a limited RNAi screen for nuclear proteins required for border cell migration, which revealed that the gene encoding Tousled-like kinase (Tlk) is required in polar cells for Upd expression without affecting polar cell fate. In the absence of Tlk, fewer border cells are recruited and motility is impaired, similar to inhibition of JAK/STAT signaling. It was further shown Tlk in polar cells is required for JAK/STAT activation in border cells. Genetic interactions further confirmed Tlk as a new regulator of Upd/JAK/STAT signaling. These findings shed light on the molecular mechanisms regulating the cooperation of motile and non-motile cells during collective invasion, a phenomenon that may also drive metastatic cancer.

Munoz-Soriano, V., Santos, D., Durupt, F. C., Casani, S. and Paricio, N. (2015). Scabrous overexpression in the eye affects R3/R4 cell fate specification and inhibits Notch signaling. Dev Dyn [Epub ahead of print]. PubMed ID: 26505171
Planar cell polarity (PCP) in the Drosophila eye is generated when immature ommatidial preclusters acquire opposite chirality in the dorsal and ventral halves of the eye imaginal disc and rotate 90 degrees towards the equator. The scabrous (sca) gene is involved in R8 differentiation and in the correct spacing of ommatidial clusters in eye imaginal discs, but it was also suggested to be required during ommatidial rotation. However, no clear relationships between sca and other genes involved in the process were established. To explore the role of Sca in PCP establishment, an RNAi-based modifier genetic screen was performed using the rough eye phenotype of sca-overexpressing flies. sca overexpression was found to mainly affect R3/R4 cell specification as it has been reported in Notch mutants. Of the 86 modifiers identified in the screen, genes encoding components of Notch signaling and proteins involved in intracellular transport were of particular interest. These and other results obtained with a reporter line of Notch activity indicate that sca overexpression antagonizes Notch signaling in the Drosophila eye, and are inconsistent with Sca being an ommatidial rotation-specific factor. Microtubule motors and other proteins involved in intracellular transport were found to be related with Sca function.

Cerny, A. C., Altendorfer, A., Schopf, K., Baltner, K., Maag, N., Sehn, E., Wolfrum, U. and Huber, A. (2015). The GTP- and phospholipid-binding protein TTD14 regulates trafficking of the TRPL ion channel in Drosophila photoreceptor cells. PLoS Genet 11: e1005578. PubMed ID: 26509977
Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels Transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. This study identified the gene mutated in trpl translocation defective 14 (ttd14) (CG30118), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane.

Thursday, November 26th

Luchtenborg, A. M., Purvanov, V., Melnik, B. S., Becker, S. and Katanaev, V. L. (2015). Mode of interaction of the Gαo subunit of heterotrimeric G proteins with the GoLoco1 motif of Drosophila Pins is determined by guanine nucleotides. Biosci Rep. PubMed ID: 26487707
Drosophila GoLoco motif-containing protein Pins is unusual in its highly efficient interaction with both GDP- and the GTP-loaded forms of the α-subunit of the heterotrimeric Go protein. This study analyzed the interactions of Gαo in its two nucleotide forms with GoLoco1 - the first of the three GoLoco domains of Pins - and the possible structures of the resulting complexes, through combination of conventional fluorescence and Forster resonance energy transfer measurements as well as through molecular modeling. The data suggest that the orientation of the GoLoco1 motif on Gαo significantly differs between the two nucleotide states of the latter. In other words, a rotation of the GoLoco1 peptide in respect with Gαo must accompany the nucleotide exchange in Gαo. The sterical hindrance requiring such a rotation likely contributes to the guanine nucleotide exchange inhibitor activity of GoLoco1 and Pins as a whole. These data have important implications for the mechanisms of Pins regulation in the process of asymmetric cell divisions.

Zhang, C., Robinson, B. S., Xu, W., Yang, L., Yao, B., Zhao, H., Byun, P. K., Jin, P., Veraksa, A. and Moberg, K. H. (2015). The ecdysone receptor coactivator Taiman links Yorkie to transcriptional control of germline stem cell factors in somatic tissue. Dev Cell 34: 168-180. PubMed ID: 26143992
The Hippo pathway is a conserved signaling cascade that modulates tissue growth. Although its core elements are well defined, factors modulating Hippo transcriptional outputs remain elusive. This study shows that components of the steroid-responsive ecdysone (Ec) pathway modulate Hippo transcriptional effects in imaginal disc cells. The Ecdysone receptor coactivator Taiman (Tai) interacts with the Hippo transcriptional coactivator Yorkie (Yki) and promotes expression of canonical Yki-responsive genes. Tai enhances Yki-driven growth, while Tai loss, or a form of Tai unable to bind Yki, suppresses Yki-driven tissue growth. This growth suppression is not correlated with impaired induction of canonical Hippo-responsive genes but with suppression of a distinct pro-growth program of Yki-induced/Tai-dependent genes, including the germline stem cell factors nanos and piwi. These data reveal Hippo/Ec pathway crosstalk in the form a Yki-Tai complex that collaboratively induces germline genes as part of a transcriptional program that is normally repressed in developing somatic epithelia.

Santabarbara-Ruiz, P., et al. (2015). ROS-induced JNK and p38 signaling is required for Unpaired cytokine activation during Drosophila regeneration. PLoS Genet 11: e1005595. PubMed ID: 26496642
Upon apoptotic stimuli, epithelial cells compensate the gaps left by dead cells by activating proliferation. This has led to the proposal that dying cells signal to surrounding living cells to maintain homeostasis. Although the nature of these signals is not clear, reactive oxygen species (ROS) could act as a signaling mechanism as they can trigger pro-inflammatory responses to protect epithelia from environmental insults. Whether ROS emerge from dead cells and what is the genetic response triggered by ROS is pivotal to understand regeneration of Drosophila imaginal discs. Cell death was genetically induced in wing imaginal discs, the production of ROS was monitored, and the signals required for repair were analyzed. Cell death was found to generate a burst of ROS that propagates to the nearby surviving cells. Propagated ROS activate p38 and induce tolerable levels of JNK. The activation of JNK and p38 results in the expression of the cytokines Unpaired (Upd), which triggers the JAK/STAT signaling pathway required for regeneration. These findings demonstrate that this ROS/JNK/p38/Upd stress responsive module restores tissue homeostasis. This module is not only activated after cell death induction but also after physical damage and reveals one of the earliest responses for imaginal disc regeneration.

Ambegaonkar, A. A. and Irvine, K. D. (2015). Coordination of planar cell polarity pathways through Spiny legs. Elife 4 [Epub ahead of print]. PubMed ID: 26505959
Morphogenesis and physiology of tissues and organs requires planar cell polarity (PCP) systems that orient and coordinate cells and their behaviors, but the relationship between PCP systems has been controversial. This study characterized how the Frizzled and Dachsous-Fat PCP systems are connected through the Spiny-legs isoform of the Prickle-Spiny-legs locus. Two different components of the Dachsous-Fat system, Dachsous and Dachs, can each independently interact with Spiny-legs and direct its localization in vivo. Through characterization of the contributions of Prickle, Spiny-legs, Dachsous, Fat, and Dachs to PCP in the Drosophila wing, eye, and abdomen, this study defined where Dachs-Spiny-legs and Dachsous-Spiny-legs interactions contribute to PCP and provides a new understanding of the orientation of polarity and the basis of PCP phenotypes. These results support the direct linkage of PCP systems through Sple in specific locales, while emphasizing that cells can be subject to and must ultimately resolve distinct, competing PCP signals.

Wednesday, November 25th

Skinner, A., Khan, S. J. and Smith-Bolton, R. K. (2015). Trithorax regulates systemic signaling during Drosophila imaginal disc regeneration. Development 142: 3500-3511. PubMed ID: 26487779
Although tissue regeneration has been studied in a variety of organisms, from Hydra to humans, many of the genes that regulate the ability of each animal to regenerate remain unknown. The larval imaginal discs of the genetically tractable model organism Drosophila melanogaster have complex patterning, well-characterized development and a high regenerative capacity, and are thus an excellent model system for studying mechanisms that regulate regeneration. To identify genes that are important for wound healing and tissue repair, a genetic screen was carried out for mutations that impair regeneration in the wing imaginal disc. Through this screen the chromatin-modification gene trithorax was identified as a key regeneration gene. This study shows that animals heterozygous for trithorax are unable to maintain activation of a developmental checkpoint that allows regeneration to occur. This defect is likely to be caused by abnormally high expression of puckered, a negative regulator of Jun N-terminal kinase (JNK) signaling, at the wound site. Insufficient JNK signaling leads to insufficient expression of an insulin-like peptide, dILP8, which is required for the developmental checkpoint. Thus, trithorax regulates regeneration signaling and capacity.

Sienski, G., Batki, J., Senti, K. A., Donertas, D., Tirian, L., Meixner, K. and Brennecke, J. (2015). Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery. Genes Dev 29: 2258-2271. PubMed ID: 26494711
The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWI-piRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. This study characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwi-mediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged Piwi-piRNA complex, it is suggested that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery.

Abramov, Y. A., Shatskikh, A. S., Maksimenko, O. G., Bonaccorsi, S., Gvozdev, V. A. and Lavrov, S. A. (2015). The differences between cis- and trans- gene inactivation caused by heterochromatin in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 26500261
Position effect variegation (PEV) is the epigenetic disruption of genes expression near the de novo formed eu-heterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. This study characterized a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous non-rearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in 500 kb region and demonstrates small non-uniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. Trans-inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. Trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2.

Erokhin, M., Elizar'ev, P., Parshikov, A., Schedl, P., Georgiev, P. and Chetverina, D. (2015). Transcriptional read-through is not sufficient to induce an epigenetic switch in the silencing activity of Polycomb response elements. Proc Natl Acad Sci U S A. PubMed ID: 26504232
In Drosophila, Polycomb (PcG) and Trithorax (TrxG) group proteins are assembled on Polycomb response elements (PREs) to maintain tissue and stage-specific patterns of gene expression. Critical to coordinating gene expression with the process of differentiation, the activity of PREs can be switched "on" and "off." When on, the PRE imposes a silenced state on the genes in the same domain that is stably inherited through multiple rounds of cell division. When the PRE is switched off, the domain is in a state permissive for gene expression that can be stably inherited. Previous studies have suggested that a burst of transcription through a PRE sequence displaces PcG proteins and provides a universal mechanism for inducing a heritable switch in PRE activity from on to off; however, the evidence favoring this model is indirect. This study has directly tested the transcriptional read-through mechanism. Contrary to previous suggestions, it was shown that transcription through the PRE is not sufficient for inducing an epigenetic switch in PRE activity. In fact, even high levels of continuous transcription through a PRE fails to dislodge the PcG proteins, nor does it remove repressive histone marks. These results indicate that other mechanisms involving adjacent DNA regulatory elements must be implicated in heritable switch of PRE activity.

Tuesday, November 24th

Bieli, D., Kanca, O., Requena, D., Hamaratoglu, F., Gohl, D., Schedl, P., Affolter, M., Slattery, M., Muller, M. and Estella, C. (2015). Establishment of a developmental compartment requires interactions between three synergistic cis-regulatory modules. PLoS Genet 11: e1005376. PubMed ID: 26468882
The subdivision of cell populations in compartments is a key event during animal development. In Drosophila, the gene apterous (ap) divides the wing imaginal disc in dorsal vs ventral cell lineages and is required for wing formation. ap function as a dorsal selector gene has been extensively studied. However, the regulation of its expression during wing development is poorly understood. This study analyzed ap transcriptional regulation at the endogenous locus and identified three cis-regulatory modules (CRMs) essential for wing development. Only when the three CRMs are combined, robust ap expression is obtained. In addition, the trans-factors that regulate these CRMs were genetically and molecularly analyzed. The results propose a three-step mechanism for the cell lineage compartment expression of ap that includes initial activation, positive autoregulation and Trithorax-mediated maintenance through separable CRMs.

Lin, Y., Sohn, C. H., Dalal, C. K., Cai, L. and Elowitz, M. B. (2015). Combinatorial gene regulation by modulation of relative pulse timing. Nature 527: 54-58. PubMed ID: 26466562
Studies of individual living cells have revealed that many transcription factors activate in dynamic, and often stochastic, pulses within the same cell. However, it has remained unclear whether cells might exploit the dynamic interaction of these pulses to control gene expression. In Saccharomyces cerevisiae, the pulsatile transcription factors Msn2 and Mig1 combinatorially regulate their target genes through modulation of their relative pulse timing. The activator Msn2 and repressor Mig1 showed pulsed activation in either a temporally overlapping or non-overlapping manner during their transient response to different inputs, with only the non-overlapping dynamics efficiently activating target gene expression. Similarly, under constant environmental conditions, where Msn2 and Mig1 exhibit sporadic pulsing, glucose concentration modulated the temporal overlap between pulses of the two factors. Together, these results reveal a time-based mode of combinatorial gene regulation. These results suggest that regulation through relative signal timing could function broadly within the signalling and regulatory systems of the cell.

Gao, G. N., Wang, M., Yang, N., Huang, Y. and Xu, R. M. (2015). Structure of Zeste-DNA complex reveals a new modality of DNA recognition by Homeodomain-like proteins. J Mol Biol [Epub ahead of print]. PubMed ID: 26478222
Drosophila Zeste is a DNA binding protein important for chromatin-targeted regulation of gene expression. It is best studied in the context of transvection-a mechanism of interallelic gene regulation involving paired chromosomes-and repression of the expression of white by Zeste mutants. Both of these functions depend on the DNA binding and self-association properties of Zeste, but the underlying structural basis remains unknown. This sudy reports the crystal structure of the DNA binding domain of Zeste in complex with a 19-bp DNA duplex containing the consensus recognition sequence motif. The structure reveals a helix-turn-helix Myb/homeodomain-like fold with the Zeste-specific insertion sequence forming a short helix and a long loop. Direct base contacts by the major groove binding helix principally account for the sequence-specific recognition, and backbone contacts via the Zeste-specific insertion are mainly responsible for the length requirement and the orientation of DNA. These structural and biochemical characterizations of the DNA binding property of Zeste uncover an altered DNA binding modality of homeodomain-like proteins, and the structural information should facilitate the unraveling of the intricate mechanism of Zeste in regulation of gene expression.

Miyagi, R., Akiyama, N., Osada, N. and Takahashi, A. (2015). Complex patterns of cis-regulatory polymorphisms in ebony underlie standing pigmentation variation in Drosophila melanogaster. Mol Ecol [Epub ahead of print]. PubMed ID: 26503353
Pigmentation traits in adult Drosophila were used to investigate how phenotypic variations of continuous ecological traits can be maintained in a natural population. First, pigmentation variation in the adult female was measured at seven different body positions in 20 strains from the Drosophila Genetic Reference Panel (DGRP) originating from a natural population in North Carolina. Next, allele-specific expression levels of four genes were quantified by amplicon sequencing. Among those genes, ebony was significantly associated with pigmentation intensity of the thoracic segment. Detailed sequence analysis of the gene regulatory regions of this gene indicated that many different functional cis-regulatory alleles are segregating in the population and that variations outside the core enhancer element could potentially play important roles in the regulation of gene expression. In contrast, sequence analysis in the core cis-regulatory region of tan indicated that SNPs within the region are significantly associated with allele-specific expression level of this gene. Collectively, the data suggest that the underlying genetic differences in the cis-regulatory regions that control intraspecific pigmentation variation can be more complex than those of interspecific pigmentation trait differences, where causal genetic changes are typically confined to modular enhancer elements.

Monday, November 23rd

Johnson, T. K., Henstridge, M. A., Herr, A., Moore, K. A., Whisstock, J. C. and Warr, C. G. (2015). Torso-like mediates extracellular accumulation of Furin-cleaved Trunk to pattern the Drosophila embryo termini. Nat Commun 6: 8759. PubMed ID: 26508274
Patterning of the Drosophila embryonic termini is achieved by localized activation of the Torso receptor by the growth factor Trunk. Governing this event is the perforin-like protein Torso-like, which is localized to the extracellular space at the embryo poles and has long been proposed to control localized proteolytic activation of Trunk. However, a protease involved in terminal patterning remains to be identified, and the role of Torso-like remains unknown. This study found that Trunk is cleaved intracellularly by Furin proteases. It was further shown that Trunk is secreted, and that levels of extracellular Trunk are greatly reduced in torso-like null mutants. On the basis of these and previous findings, it is suggested that Torso-like functions to mediate secretion of Trunk, thus providing the mechanism for spatially restricted activation of Torso. The data represent an alternative mechanism for the spatial control of receptor signalling, and define a different role for perforin-like proteins in eukaryotes.

Tamada, M. and Zallen, J. A. (2015). Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling. Dev Cell 35: 151-161. PubMed ID: 26506305
Cells display dynamic and diverse morphologies during development, but the strategies by which differentiated tissues achieve precise shapes and patterns are not well understood. This study identified a developmental program that generates a highly ordered square cell grid in the Drosophila embryo through sequential and spatially regulated cell alignment, oriented cell division, and apicobasal cell elongation. The basic leucine zipper transcriptional regulator Cnc is necessary and sufficient to produce a square cell grid in the presence of a midline signal provided by the EGF receptor ligand Spitz. Spitz orients cell divisions through a Pins/LGN-dependent spindle-positioning mechanism and controls cell shape and alignment through a transcriptional pathway that requires the Pointed ETS domain protein. These results identify a strategy for producing ordered square cell packing configurations in epithelia and reveal a molecular mechanism by which organized tissue structure is generated through spatiotemporally regulated responses to EGF receptor activation.

Crews, S. M., McCleery, W. T. and Hutson, M. S. (2015). Pathway to a phenocopy: Heat stress effects in early embryogenesis. Dev Dyn [Epub ahead of print]. PubMed ID: 26498920
Heat shocks applied at the onset of gastrulation in early Drosophila embryos frequently lead to phenocopies of U-shaped mutants - having characteristic failures in the late morphogenetic processes of germband retraction and dorsal closure. The pathway from non-specific heat stress to phenocopied abnormalities is unknown. Drosophila embryos subjected to 30-min, 38- degrees C heat shocks at gastrulation appear to recover and restart morphogenesis. Post-heat-shock development appears normal, albeit slower, until a large fraction of embryos develop amnioserosa holes (diameters > 100 microm). These holes are positively correlated with terminal U-shaped phenocopies. They initiate between amnioserosa cells and open over tens of minutes by evading normal wound healing responses. They are not caused by tissue-wide increases in mechanical stress or decreases in cell-cell adhesion, but instead appear to initiate from isolated apoptosis of amnioserosa cells. It is concluded that the pathway from heat shock to U-shaped phenocopies involves the opening of one or more large holes in the amnioserosa that compromise its structural integrity and lead to failures in morphogenetic processes that rely on amnioserosa-generated tensile forces. The proposed mechanism by which heat shock leads to hole initiation and expansion is heterochonicity - i.e., disruption of morphogenetic coordination between embryonic and extra-embryonic cell types.

Rauzi, M., Krzic, U., Saunders, T. E., Krajnc, M., Ziherl, P., Hufnagel, L. and Leptin, M. (2015). Embryo-scale tissue mechanics during Drosophila gastrulation movements. Nat Commun 6: 8677. PubMed ID: 26497898
Morphogenesis of an organism requires the development of its parts to be coordinated in time and space. While past studies concentrated on defined cell populations, a synthetic view of the coordination of these events in a whole organism is needed for a full understanding. Drosophila gastrulation begins with the embryo forming a ventral furrow, which is eventually internalized. It is not understood how the rest of the embryo participates in this process. This study used multiview selective plane illumination microscopy coupled with infrared laser manipulation and mutant analysis to dissect embryo-scale cell interactions during early gastrulation. Lateral cells have a denser medial-apical actomyosin network and shift ventrally as a compact cohort, whereas dorsal cells become stretched. The behaviour of these cells affects furrow internalization. A computational model predicts different mechanical properties associated with tissue behaviour: lateral cells are stiff, whereas dorsal cells are soft. Experimental analysis confirms these properties in vivo.

Sunday, November 22nd

Yadav, S. and Tapadia, M.G. (2015). Expression of polyQ aggregates in Malpighian tubules leads to degeneration in Drosophila melanogaster. Dev Biol [Epub ahead of print]. PubMed ID: 26517966
Polyglutamine (polyQ) disorders are caused by expanded CAG (Glutamine) repeats in neurons in the brain. The expanded repeats are also expressed in the non-neuronal cells, however, their contribution to disease pathogenesis is not very well studied. This study expressed a stretch of 127 Glutamine repeats in Malpighian tubules (MTs) of Drosophila melanogaster as these tissues do not undergo ecdysone induced histolysis during larval to pupal transition at metamorphosis. Progressive degeneration, which is the hallmark of neurodegeneration was also observed in MTs. The mutant protein forms inclusion bodies in the nucleus resulting in expansion of the nucleus and affect chromatin organization which appear loose and open, eventually resulting in DNA fragmentation and blebbing. A virtual absence of tubule lumen was observed followed by functional abnormalities. As development progressed, severe abnormalities affecting pupal epithelial morphogenesis processes were observed resulting in complete lethality. Distribution of heterogeneous RNA binding protein (hnRNP), HRB87F, Wnt/wingless and JNK signaling and expression of Relish was also found to be affected. Expression of multi-drug resistance genes following polyQ expression was up regulated. The study gives an insight into the effects of polyQ aggregates in non-neuronal tissues.

Merzetti, E. M. and Staveley, B. E. (2015). spargel, the PGC-1alpha homologue, in models of Parkinson disease in Drosophila melanogaster. BMC Neurosci 16: 70. PubMed ID: 26502946
Parkinson disease (PD) is a progressive neurodegenerative disorder that arises from a decrease of available dopamine in the striatum of the brain resulting from the breakdown and death of dopaminergic (DA) neurons. PGC-1alpha has been characterized as a regulator of mitochondria biogenesis, insulin receptor signalling and energy metabolism, mutation of this gene has been linked to early onset forms of PD. The sole Drosophila homologue of this gene family, spargel (srl), has been shown to function in similar pathways of mitochondrial upkeep and biogenesis. Directed expression of srl-RNAi in the Drosophila eye causes abnormal ommatidia and bristle formation while eye specific expression of srl-EY does not produce the minor rough eye phenotype associated with high temperature GMR-Gal4 expression. Ddc-Gal4 mediated tissue specific expression of srl transgene constructs in Drosophila DA neurons causes altered lifespan and climbing ability. Expression of a srl-RNAi causes an increase in mean lifespan but a decrease in overall loco-motor ability while induced expression of srl-EY causes a severe decrease in mean lifespan and a decrease in loco-motor ability. The reduced lifespan and climbing ability associated with a tissue specific expression of srl in DA neurons provides a new model of PD in D. melanogaster which may be used to identify novel therapeutic approaches to human disease treatment and prevention.

Cragnaz, L., Klima, R., De Conti, L., Romano, G., Feiguin, F., Buratti, E., Baralle, M. and Baralle, F. E. (2015). An age-related reduction of brain TBPH/TDP-43 levels precedes the onset of locomotion defects in a Drosophila ALS model. Neuroscience 311: 415-421. PubMed ID: 26518462
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The average age of onset of both sporadic and familial cases is 50-60 years of age. The presence of cytoplasmic inclusions of the RNA-binding protein TAR DNA-binding protein-43 (TDP-43) in the affected neurons is seen in 95% of the ALS cases, which results in TDP-43 nuclear clearance and loss of function. The Drosophila melanogaster ortholog of TDP-43 (TBPH) shares many characteristics with the human protein. Using a TDP-43 aggregation inducer previously developed in human cells, a transgenic fly was created that shows an adult locomotive defect. Phenotype onset correlates with a physiologically age-related drop of TDP-43/TBPH mRNA and protein levels, seen both in mice and flies. Artificial reduction of mRNA levels, in vivo, anticipates the locomotion defect to the larval stage. This study links, for the first time, aggregation and the age-related, evolutionary conserved reduction of TDP-43/TBPH levels with the onset of an ALS-like locomotion defect in a Drosophila model. A similar process might trigger the human disease.

Di Salvio, M., et al. (2015). Pur-α functionally interacts with FUS carrying ALS-associated mutations. Cell Death Dis 6: e1943. PubMed ID: 26492376.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to motor neuron loss. Fused in sarcoma (FUS) protein carrying ALS-associated mutations localizes to stress granules and causes their coalescence into larger aggregates. This study shows that Pur-α physically interacts with mutated FUS in an RNA-dependent manner. Pur-α colocalizes with FUS carrying mutations in stress granules of motoneuronal cells differentiated from induced pluripotent stem cells and that are derived from ALS patients. Both Pur-α and mutated FUS upregulate phosphorylation of the translation initiation factor EIF-2α and consistently inhibit global protein synthesis. In vivo expression of Pur-α in different Drosophila tissues significantly exacerbates the neurodegeneration caused by mutated FUS. Conversely, the downregulation of Pur-α in neurons expressing mutated FUS significatively improves fly climbing activity. All these findings suggest that Pur-α, through the control of mRNA translation, might be involved in the pathogenesis of ALS associated with the mutation of FUS, and that an alteration of protein synthesis may be directly implicated in the disease. Finally, in vivo RNAi-mediated ablation of Pur-α produced locomotion defects in Drosophila, indicating a pivotal role for this protein in the motoneuronal function.

Saturday, November 21st

Travers, L. M., Garcia-Gonzalez, F. and Simmons, L. W. (2015). Live fast die young life history in females: evolutionary trade-off between early life mating and lifespan in female Drosophila melanogaster. Sci Rep 5: 15469. PubMed ID: 26482533
The trade-off between survival and reproduction is fundamental to life history theory. Sexual selection is expected to favour a 'live fast die young' life history pattern in males due to increased risk of extrinsic mortality associated with obtaining mates. Sexual conflict may also drive a genetic trade-off between reproduction and lifespan in females. This study found significant additive genetic variance in longevity independent of lifetime mating frequency, and in early life mating frequency. There was significant negative genetic covariance between these traits indicating that females from families characterized by high levels of multiple mating early in life die sooner than females that engage in less intense early life mating. Thus, despite heritable variation in both traits, their independent evolution is constrained by an evolutionary trade-off. These findings indicate that, in addition to the well-known male-driven direct costs of mating on female lifespan (mediated by male harassment and harmful effects of seminal fluids), females with a genetic propensity to mate multiply live shorter lives. The potential role of sexual conflict in driving the evolutionary trade-off between reproduction and lifespan in Drosophila is discussed. More generally, the data show that, like males, females can exhibit a live fast die young life history strategy.

Paris, M., Villalta, J. E., Eisen, M. B. and Lott, S. E. (2015). Sex bias and maternal contribution to gene expression divergence in Drosophila blastoderm embryos. PLoS Genet 11: e1005592. PubMed ID: 26485701
Early embryogenesis is a unique developmental stage where genetic control of development is handed off from mother to zygote. Yet the contribution of this transition to the evolution of gene expression is poorly understood. This study examined two aspects of gene expression specific to early embryogenesis in Drosophila: sex-biased gene expression prior to the onset of canonical X chromosomal dosage compensation, and the contribution of maternally supplied mRNAs. mRNAs were sequenced from individual unfertilized eggs and precisely staged and sexed blastoderm embryos, and levels between D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis were compared. First, it was found that mRNA content is highly conserved for a given stage and that studies relying on pooled embryos likely systematically overstate the degree of gene expression divergence. Unlike studies done on larvae and adults where most species show a larger proportion of genes with male-biased expression, this study found that transcripts in Drosophila embryos are largely female-biased in all species, likely due to incomplete dosage compensation prior to the activation of the canonical dosage compensation mechanism. The divergence of sex-biased gene expression across species is observed to be often due to lineage-specific decrease of expression; the most drastic example of which is the overall reduction of male expression from the neo-X chromosome in D. pseudoobscura, leading to a pervasive female-bias on this chromosome. No evidence was seen for a faster evolution of expression on the X chromosome in embryos (no "faster-X" effect), unlike in adults, and contrary to a previous study on pooled non-sexed embryos. Finally, it was found that most genes are conserved in regard to their maternal or zygotic origin of transcription, and evidence is presented that differences in maternal contribution to the blastoderm transcript pool may be due to species-specific divergence of transcript degradation rates.

Zhang, Z. and Presgraves, D. C. (2015). Drosophila X-linked genes have lower translation rates than autosomal genes. Mol Biol Evol [Epub ahead of print]. PubMed ID: 26486873
In Drosophila, X-linked and autosomal genes achieve comparable expression at the mRNA level. Whether comparable X-autosome gene expression is realized at the translational and, ultimately, the protein levels is however unknown. Previous studies suggest the possibility of higher translation rates for X-linked genes owing to stronger usage of preferred codons. This study used public ribosome profiling data from Drosophila melanogaster to infer translation rates on the X chromosome versus the autosomes. X-linked genes were found to have consistently lower ribosome densities than autosomal genes in S2 cells, early embryos, eggs, and mature oocytes. Surprisingly, the lower ribosome densities of X-linked genes are not consistent with faster translation elongation but instead imply slower translation initiation. In particular, X-linked genes have sequence features known to slow translation initiation such as stronger mRNA structure near start codons and longer 5' UTRs. Comparison to outgroup species suggests that stronger mRNA structure is an evolved feature of Drosophila X chromosomes. Finally, the magnitude of the X-autosome difference in ribosome densities was found to be smaller for genes encoding members of protein complexes, suggesting that stoichiometry constrains the evolution of translation rates. In sum, these analyses suggest that Drosophila X-linked genes have evolved lower translation rates than autosomal genes despite stronger usage of preferred codons.

Castellano, D., Coronado-Zamora, M., Campos, J. L., Barbadilla, A. and Eyre-Walker, A. (2015). Adaptive evolution is substantially impeded by Hill-Robertson interference in Drosophila. Mol Biol Evol [Epub ahead of print]. PubMed ID: 26494843
Hill-Robertson interference (HRi) is expected to reduce the efficiency of natural selection when two or more linked selected sites do not segregate freely, but no attempt has been done so far to quantify the overall impact of HRi on the rate of adaptive evolution for any given genome. This work estimates how much HRi impedes the rate of adaptive evolution in the coding genome of D. melanogaster. A dataset of 6,141 autosomal protein coding genes was compiled from Drosophila, from which polymorphism levels in D. melanogaster and divergence out to D. yakuba were estimated. The rate of adaptive evolution was calculated using a derivative of the McDonald-Kreitman test that controls for slightly deleterious mutations. The rate of adaptive amino acid substitution at a given position of the genome was found to be positively correlated to both the rate of recombination and the mutation rate, and negatively correlated to the gene density of the region. These correlations are robust to controlling for each other, for synonymous codon bias and for gene functions related to immune response and testes. HRi diminishes the rate of adaptive evolution by ~27%. Interestingly, genes with low mutation rates embedded in gene poor regions lose ~17% of their adaptive substitutions while genes with high mutation rates embedded in gene rich regions lose ~60%. It is concluded that HRi hampers the rate of adaptive evolution in Drosophila and that the variation in recombination, mutation and gene density along the genome affects the HRi effect.

Friday, November 20th

Shaposhnikov, M., Proshkina, E., Shilova, L., Zhavoronkov, A. and Moskalev, A. (2015). Lifespan and stress resistance in Drosophila with overexpressed DNA repair genes. Sci Rep 5: 15299. PubMed ID: 26477511
DNA repair declines with age and correlates with longevity in many animal species. This study investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster.

Brinzer, R. A., Henderson, L., Marchiondo, A. A., Woods, D. J., Davies, S. A. and Dow, J. A. (2015). Metabolomic profiling of permethrin-treated Drosophila melanogaster identifies a role for tryptophan catabolism in insecticide survival. Insect Biochem Mol Biol. PubMed ID: 26474926
Insecticides and associated synergists are rapidly losing efficacy in target insect pest populations making the discovery of alternatives a priority. To discover novel targets for permethrin synergists, metabolomics was performed on permethrin-treated Drosophila melanogaster. Changes were observed in several metabolic pathways including those for amino acids, glycogen, glycolysis, energy, nitrogen, NAD+, purine, pyrimidine, lipids and carnitine. Markers for acidosis, ammonia stress, oxidative stress and detoxification responses were also observed. Many of these changes had not been previously characterized after permethrin exposure. From the altered pathways, tryptophan catabolism was selected for further investigation. The knockdown of some tryptophan catabolism genes (vermilion, cinnabar and CG6950) in the whole fly and in specific tissues including fat body, midgut and Malpighian tubules using targeted RNAi resulted in altered survival phenotypes against acute topical permethrin exposure. The knockdown of vermilion, cinnabar and CG6950 in the whole fly also altered survival phenotypes against chronic oral permethrin, fenvalerate, DDT, chlorpyriphos and hydramethylnon exposure. Thus tryptophan catabolism has a previously uncharacterized role in defence against insecticides, and shows that metabolomics is a powerful tool for target identification in pesticide research.

Moghadam, N. N., Holmstrup, M., Manenti, T. and Loeschcke, V. (2015). Phospholipid fatty acid composition linking larval-density to lifespan of adult Drosophila melanogaster. Exp Gerontol 72: 177-183. PubMed ID: 26481768
Pre-adult density-associated alterations in the composition of storage lipids may affect the cell membrane fatty acid profile (mainly phospholipids), membrane integrity, and cell function. The present study evaluated the impact of pre-adult density conditions, sex, and the selection regime on the composition of phospholipid fatty acids and lifespan of Drosophila melanogaster. The phospholipid profile of adult flies developed under larval crowding contained a higher proportion of polyunsaturated fatty acids, lower proportion of monounsaturated fatty acids, and greater risk of peroxidation. There was also a negative correlation between the peroxidation index (PI) and longevity. The longevity-selected females showed a lower PI compared with control lines under both densities. The present results indicate that pre-adult density may play a significant role in the lifespan of adult flies by altering the composition of phospholipids and shaping cell membrane bilayers with different susceptibilities to peroxidation.

Shaukat, Z., Liu, D., Choo, A., Hussain, R., O'Keefe, L., Richards, R., Saint, R. and Gregory, S. L. (2015). Chromosomal instability causes sensitivity to metabolic stress. Oncogene 34: 4044-4055. PubMed ID: 25347746
Chromosomal INstability (CIN), a hallmark of cancer, refers to cells with an increased rate of gain or loss of whole chromosomes or chromosome parts. CIN is linked to the progression of tumors with poor clinical outcomes such as drug resistance. CIN can give tumors the diversity to resist therapy, but it comes at the cost of significant stress to tumor cells. To tolerate this, cancer cells must modify their energy use to provide adaptation against genetic changes as well as to promote their survival and growth. This study has demonstrated that CIN induction causes sensitivity to metabolic stress. Mild metabolic disruption that does not affect normal cells can lead to high levels of oxidative stress and subsequent cell death in CIN cells because they are already managing elevated stress levels. Altered metabolism is a differential characteristic of cancer cells, so identification of key regulators that can exploit these changes to cause cell death may provide cancer-specific potential drug targets, especially for advanced cancers that exhibit CIN.

Thursday, November 19th

Chen, J. V., Kao, L. R., Jana, S. C., Sivan-Loukianova, E., Mendonca, S., Cabrera, O. A., Singh, P., Cabernard, C., Eberl, D. F., Bettencourt-Dias, M. and Megraw, T. L. (2015). Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila.J Cell Biol 211: 435-453. PubMed ID: 26483560
Cilia are essential for cell signaling and sensory perception. In many cell types, a cytoskeletal structure called the ciliary rootlet links the cilium to the cell body. Previous studies indicated that rootlets support the long-term stability of some cilia. This study reports that Drosophila melanogaster Rootletin (Root), the sole orthologue of the mammalian paralogs Rootletin and C-Nap1, assembles into rootlets of diverse lengths among sensory neuron subtypes. Root mutant neurons lack rootlets and have dramatically impaired sensory function, resulting in behavior defects associated with mechanosensation and chemosensation. Root is required for cohesion of basal bodies, but the cilium structure appears normal in Root mutant neurons. Normal rootlet assembly requires centrioles. The N terminus of Root contains a conserved domain and is essential for Root function in vivo. Ectopically expressed Root resides at the base of mother centrioles in spermatocytes and localizes asymmetrically to mother centrosomes in neuroblasts, both requiring Bld10, a basal body protein with varied functions.

Wang, X., Zhang, M. W., Kim, J. H., Macara, A. M., Sterne, G., Yang, T. and Ye, B. (2015). The Kruppel-like factor Dar1 determines multipolar neuron morphology. J Neurosci 35: 1425259. PubMed ID: 26490864
Neurons typically assume multipolar, bipolar, or unipolar morphologies. Little is known about the mechanisms underlying the development of these basic morphological types. This study shows that the Kruppel-like transcription factor Dar1 determines the multipolar morphology of postmitotic neurons in Drosophila. Dar1 is specifically expressed in multipolar neurons and loss of dar1 gradually converts multipolar neurons into the bipolar or unipolar morphology without changing neuronal identity. Conversely, misexpression of Dar1 or its mammalian homolog in unipolar and bipolar neurons causes them to assume multipolar morphologies. Dar1 regulates the expression of several dynein genes and nuclear distribution protein C (nudC), which is an essential component of a specialized dynein complex that positions the nucleus in a cell. These genes were shown to be required for Dar1-induced multipolar neuron morphology. Dar1 likely functions as a terminal selector gene for the basic layout of neuron morphology by regulating both dendrite extension and the dendrite-nucleus coupling.

Cagin, U., Duncan, O. F., Gatt, A. P., Dionne, M. S., Sweeney, S. T. and Bateman, J. M. (2015). Mitochondrial retrograde signaling regulates neuronal function. Proc Natl Acad Sci U S A 112: E6000-6009. PubMed ID: 26489648
Mitochondria are key regulators of cellular homeostasis, and mitochondrial dysfunction is strongly linked to neurodegenerative diseases, including Alzheimer's and Parkinson's. Mitochondria communicate their bioenergetic status to the cell via mitochondrial retrograde signaling. To investigate the role of mitochondrial retrograde signaling in neurons, we induced mitochondrial dysfunction in the Drosophila nervous system. Neuronal mitochondrial dysfunction causes reduced viability, defects in neuronal function, decreased redox potential, and reduced numbers of presynaptic mitochondria and active zones. Neuronal mitochondrial dysfunction stimulates a retrograde signaling response that controls the expression of several hundred nuclear genes. Drosophila hypoxia inducible factor alpha (HIFalpha) ortholog Similar (Sima) regulates the expression of several of these retrograde genes, suggesting that Sima mediates mitochondrial retrograde signaling. Remarkably, knockdown of Sima restores neuronal function without affecting the primary mitochondrial defect, demonstrating that mitochondrial retrograde signaling is partly responsible for neuronal dysfunction. Sima knockdown also restores function in a Drosophila model of the mitochondrial disease Leigh syndrome and in a Drosophila model of familial Parkinson's disease. Thus, mitochondrial retrograde signaling regulates neuronal activity and can be manipulated to enhance neuronal function, despite mitochondrial impairment.

McParland, A. L., Follansbee, T. L., Vesenka, G. D., Panaitiu, A. E. and Ganter, G. K. (2015). Steroid receptor isoform expression in Drosophila nociceptor neurons is required for normal dendritic arbor and sensitivity. PLoS One 10: e0140785. PubMed ID: 26495837
Steroid hormones organize many aspects of development, including that of the nervous system. Steroids also play neuromodulatory and other activational roles, including regulation of sensitivity to painful stimuli in mammals. In Drosophila, ecdysteroids are the only steroid hormones, and therefore the fly represents a simplified model system in which to explore mechanisms of steroid neuromodulation of nociception. In this report, evidence is presented that ecdysteroids, acting through two isoforms of their nuclear ecdysone receptor (EcR), modulate sensitivity to noxious thermal and mechanical stimuli in the fly larva. EcRA and EcRB1 are expressed by third instar larvae in the primary nociceptor neurons, known as the class IV multidendritic neurons. Suppression of EcRA by RNA interference in these cells leads to hyposensitivity to noxious stimulation. Suppression of EcRB1 leads to reduction of dendritic branching and length of nociceptor neurons. Specific isoforms of the ecdysone receptor play critical cell autonomous roles in modulating the sensitivity of nociceptor neurons and may indicate human orthologs that represent targets for novel analgesic drugs.

Wednesday, November 18th

Chen, C. H., Luhur, A. and Sokol, N. (2015). Lin-28 promotes symmetric stem cell division and drives adaptive growth in the adult Drosophila intestine. Development 142: 3478-3487. PubMed ID: 26487778
Stem cells switch between asymmetric and symmetric division to expand in number as tissues grow during development and in response to environmental changes. The stem cell intrinsic proteins controlling this switch are largely unknown, but one candidate is the Lin-28 pluripotency factor. A conserved RNA-binding protein that is downregulated in most animals as they develop from embryos to adults, Lin-28 persists in populations of adult stem cells. Its function in these cells has not been previously characterized. This study reports that Lin-28 is highly enriched in adult intestinal stem cells in the Drosophila intestine. lin-28 null mutants are homozygous viable but display defects in this population of cells, which fail to undergo a characteristic food-triggered expansion in number and have reduced rates of symmetric division as well as reduced insulin signaling. Immunoprecipitation of Lin-28-bound mRNAs identified Insulin-like Receptor (InR), forced expression of which completely rescues lin-28-associated defects in intestinal stem cell number and division pattern. Furthermore, this stem cell activity of lin-28 is independent of one well-known lin-28 target, the microRNA let-7, which has limited expression in the intestinal epithelium. These results identify Lin-28 as a stem cell intrinsic factor that boosts insulin signaling in intestinal progenitor cells and promotes their symmetric division in response to nutrients, defining a mechanism through which Lin-28 controls the adult stem cell division patterns that underlie tissue homeostasis and regeneration.

Park, S. Y., Stultz, B. G. and Hursh, D. A. (2015). Dual role of Jun N-terminal kinase activity in bone morphogenetic protein-mediated Drosophila ventral head development. Genetics [Epub ahead of print]. PubMed ID: 26500262
decapentaplegic (dpp) controls ventral head morphogenesis by expression in the eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. Palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the JNK pathway but in opposite ways. Slight reduction of both Jun N-terminal Kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal Kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal Kinase activity. It is concluded that formation of sensory vibrissae, rostral membrane and gena tissue requires the action of JNK in peripodial cells, while excessive Jun N-terminal Kinase signaling in these same cells inhibits the formation of maxillary palps.

Richard, M. and Hoch, M. (2015). Drosophila eye size is determined by Innexin 2-dependent Decapentaplegic signalling. Dev Biol [Epub ahead of print]. PubMed ID: 26455410
Organogenesis relies on specific genetic and molecular programmes, which orchestrate growth and cellular differentiation over developmental time. This is particularly important during Drosophila eye development in which cell-cell inductive events and long-range signalling have to be integrated to regulate proper cell proliferation, differentiation and morphogenesis. How these processes are coordinated is still not very well understood. This study identified the gap junction protein Innexin2 (Inx2) as an important regulator of eye development. Depleting inx2 during eye development reduces eye size whereas elevating inx2 levels increases eye size. Loss- and gain-of-function experiments demonstrate that inx2 is required functionally in larval eye disc cells where it localises apico-laterally. inx2 regulates disc cell proliferation as well as morphogenetic furrow movement and as a result the amount of differentiated photoreceptors. inx2 interacts genetically with the Dpp pathway, and proper activation of the Dpp pathway transducer Mad at the furrow and expression of Dpp receptors Thickveins and Punt in the anterior disc compartment were found to require inx2. It was further shown that inx2 is required for the transcriptional activation of dpp and punt in the eye disc. Our results highlight the crucial role of gap junction proteins in regulating morphogen-dependent organ size determination.

Li, Q., Barish, S., Okuwa, S. and Volkan, P. C. (2015). Examination of endogenous Rotund expression and function in developing Drosophila olfactory system using CRISPR-Cas9 mediated protein tagging. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26497147
The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. This study added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila in order to investigate its sub-cellular localization and the genes that it regulates. Previously a reporter construct was used to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. This study shows that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, the expression pattern of the endogenous isoform-specific tags were mapped in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, a direct binding was shown of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.

Tuesday, November 17th

Deady, L. D. and Sun, J. (2015). A follicle rupture assay reveals an essential role for follicular adrenergic signaling in Drosophila ovulation. PLoS Genet 11: e1005604. PubMed ID: 26473732
Ovulation is essential for the propagation of the species and involves a proteolytic degradation of the follicle wall for the release of the fertilizable oocyte. However, the precise mechanisms for regulating these proteolytic events are largely unknown. There are several parallels between Drosophila and mammalian ovulation at both the cellular and molecular levels. During ovulation in Drosophila, posterior follicle cells surrounding a mature oocyte are selectively degraded and the residual follicle cells remain in the ovary to form a corpus luteum after follicle rupture. Like in mammals, this rupturing process also depends on matrix metalloproteinase 2 (Mmp2) activity localized at the posterior end of mature follicles, where oocytes exit. This study shows that Mmp2 activity is regulated by the octopaminergic signaling in mature follicle cells. Exogenous octopamine (OA; equivalent to norepinephrine, NE) is sufficient to induce follicle rupture when isolated mature follicles are cultured ex vivo, in the absence of the oviduct or ovarian muscle sheath. Knocking down the alpha-like adrenergic receptor Oamb (Octoampine receptor in mushroom bodies) in mature follicle cells prevents OA-induced follicle rupture ex vivo and ovulation in vivo. Follicular OA-Oamb signaling induces Mmp2 enzymatic activation but not Mmp2 protein expression, likely via intracellular Ca2+ as the second messenger. This work develops a novel ex vivo follicle rupture assay and demonstrates the role for follicular adrenergic signaling in Mmp2 activation and ovulation in Drosophila, which is likely conserved in other species.

Weinberg-Shukron, A., et al. (2015). A mutation in the nucleoporin-107 gene causes XX gonadal dysgenesis. J Clin Invest 125: 4295-4304. PubMed ID: 26485283
Ovarian development and maintenance are poorly understood; however, diseases that affect these processes can offer insights into the underlying mechanisms. XX female gonadal dysgenesis (XX-GD) is a rare, genetically heterogeneous disorder that is characterized by underdeveloped, dysfunctional ovaries, with subsequent lack of spontaneous pubertal development, primary amenorrhea, uterine hypoplasia, and hypergonadotropic hypogonadism. This study reports an extended consanguineous family of Palestinian origin, in which 4 females exhibited XX-GD. Using homozygosity mapping and whole-exome sequencing, a recessive missense mutation was identified in nucleoporin-107 (NUP107, c.1339G>A, p.D447N). This mutation segregated with the XX-GD phenotype and was not present in available databases or in 150 healthy ethnically matched controls. NUP107 is a component of the nuclear pore complex, and the NUP107-associated protein SEH1 is required for oogenesis in Drosophila. In Drosophila, Nup107 knockdown in somatic gonadal cells resulted in female sterility, whereas males were fully fertile. Transgenic rescue of Drosophila females bearing the Nup107D364N mutation, which corresponds to the human NUP107 (p.D447N), resulted in almost complete sterility, with a marked reduction in progeny, morphologically aberrant eggshells, and disintegrating egg chambers, indicating defective oogenesis. These results indicate a pivotal role for NUP107 in ovarian development and suggest that nucleoporin defects may play a role in milder and more common conditions such as premature ovarian failure.

Ben-David, G., Miller, E. and Steinhauer, J. (2015). Spermatid individualization is sensitive to temperature and fatty acid metabolism. Spermatogenesis 5: e1006089. PubMed ID: 26413411
Fatty acids are precursors of potent lipid signaling molecules. They are stored in membrane phospholipids and released by phospholipase A2 (PLA2). Lysophospholipid acyltransferases (ATs) oppose PLA2 by re-esterifying fatty acids into phospholipids, in a biochemical pathway known as the Lands Cycle. Drosophila Lands Cycle ATs oys and nes, as well as 7 predicted PLA2 genes, are expressed in the male reproductive tract. Oys and Nes are required for spermatid individualization. Individualization, which occurs after terminal differentiation, invests each spermatid in its own plasma membrane and removes the bulk of the cytoplasmic contents. This study developed a quantitative assay to measure individualization defects. Individualization is demonstrated to be sensitive to temperature and age but not to diet. Mutation of the cyclooxygenase Pxt, which metabolizes fatty acids to prostaglandins, also leads to individualization defects. In contrast, modulating phospholipid levels by mutation of the phosphatidylcholine lipase Swiss cheese (Sws) or the ethanolamine kinase Easily shocked (Eas) does not perturb individualization, nor does Sws overexpression. These results suggest that fatty acid derived signals such as prostaglandins, whose abundance is regulated by the Lands Cycle, are important regulators of spermatogenesis.

Tarayrah, L., Li, Y., Gan, Q. and Chen, X. (2015). Epigenetic regulator Lid maintains germline stem cells through regulating JAK-STAT signaling pathway activity. Biol Open. PubMed ID: 26490676
Signaling pathways and epigenetic mechanisms have both been shown to play essential roles in regulating stem cell activity. While the role of either mechanism in this regulation is well established in multiple stem cell lineages, how the two mechanisms interact to regulate stem cell activity is not as well understood. This study reportd that in the Drosophila testis, an H3K4me3-specific histone demethylase encoded by little imaginal discs (lid) maintains germline stem cell (GSC) mitotic index and prevents GSC premature differentiation. Lid is required in germ cells for proper expression of the Stat92E transcription factor, the downstream effector of the JAK-STAT signaling pathway. These findings support a germ cell autonomous role for the JAK-STAT pathway in maintaining GSCs and place Lid as an upstream regulator of this pathway. This study provides new insights into the biological functions of a histone demethylase in vivo and sheds light on the interaction between epigenetic mechanisms and signaling pathways in regulating stem cell activities.

Monday, November 16th

Xie, J., Wooten, M., Tran, V., Chen, B.C., Pozmanter, C., Simbolon, C., Betzig, E. and Chen, X. (2015). Histone H3 threonine phosphorylation regulates asymmetric histone inheritance in the Drosophila male germline. Cell [Epub ahead of print]. PubMed ID: 26522592
A long-standing question concerns how stem cells maintain their identity through multiple divisions. It has been reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. This study shows that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity.

Gaykalova, D. A., Kulaeva, O. I., Volokh, O., Shaytan, A. K., Hsieh, F. K., Kirpichnikov, M. P., Sokolova, O. S. and Studitsky, V. M. (2015). Structural analysis of nucleosomal barrier to transcription. Proc Natl Acad Sci U S A 112: E5787-5795. PubMed ID: 26460019
Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. This study shows that both DNA-histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone-histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA-protein and protein-protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed.

Koya, S. K. and Meller, V. H. (2015). Modulation of heterochromatin by male specific lethal proteins and roX RNA in Drosophila melanogaster males. PLoS One 10: e0140259. PubMed ID: 26468879
The ribonucleoprotein Male Specific Lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila males. Beginning at 3 h of development the MSL complex binds transcribed X-linked genes and modifies chromatin. A subset of MSL complex proteins, including MSL1 and MSL3, is also necessary for full expression of autosomal heterochromatic genes in males, but not females. Loss of the non-coding roX RNAs, essential components of the MSL complex, lowers the expression of heterochromatic genes and suppresses position effect variegation (PEV) only in males, revealing a sex-limited disruption of heterochromatin. MLE, but not Jil-1 kinase, was found to contribute to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, a panel of roX1 transgenes and deletions was tested; the X chromosome and heterochromatin functions were found to be separable by some mutations. Widespread autosomal binding of MSL3 occurs before and after localization of the MSL complex to the X chromosome at 3 h AEL. Autosomal MSL3 binding was dependent on MSL1, supporting the idea that a subset of MSL proteins associates with chromatin throughout the genome during early development. It is postulated that this binding may contribute to the sex-specific differences in heterochromatin that have been noted.

Golovnin, A., Melnikova, L., Shapovalov, I., Kostyuchenko, M. and Georgiev, P. (2015). EAST organizes Drosophila insulator proteins in the interchromosomal nuclear compartment and modulates CP190 binding to chromatin. PLoS One 10: e0140991. PubMed ID: 26489095
Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. This study has shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.

Sunday, November 15th

Kwong, P. N., Chambers, M., Vashisht, A. A., Turki-Judeh, W., Yau, T. Y., Wohlschlegel, J. A. and Courey, A. J. (2015). The central region of the Drosophila co-repressor Groucho as a regulatory hub. J Biol Chem [Epub ahead of print]. PubMed ID: 26483546
Groucho (Gro) is a Drosophila co-repressor that regulates the expression of a large number of genes, many of which are involved in developmental control. This study has identified multiple embryonic Gro-interacting proteins. The interactors include protein complexes involved in chromosome organization, mRNA processing, and signaling. Further investigation of the interacting proteins using a reporter assay showed that many of them modulate Gro-mediated repression either positively or negatively. The positive regulators include components of the spliceosomal subcomplex U1 small nuclear ribonucleoprotein (U1 snRNP). A co-immunoprecipitation experiment confirms this finding and suggests that a sizable fraction of nuclear U1 snRNP is associated with Gro. The use of RNA-seq to analyze the gene expression profile of cells subjected to knockdown of Gro or snRNP-U1-C (a component of U1 snRNP) showed a significant overlap between genes regulated by these two factors. Furthermore, comparison of our RNA-seq data to Gro and Pol II ChIP data led to a number of insights including the finding that Gro-repressed genes are enriched for promoter proximal Pol II. It is concluded that the Gro central domains mediate multiple interactions required for repression thus functioning as a regulatory hub. Furthermore, interactions with the spliceosome may contribute to repression by Gro.

Hardie, R. C., Liu, C. H., Randall, A. S. and Sengupta, S. (2015). In vivo tracking of phosphoinositides in Drosophila photoreceptors. J Cell Sci [Epub ahead of print]. PubMed ID: 26483384
In order to monitor phosphoinositide turnover during phospholipase C (PLC) mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, TbR332H (mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for PtdIns(4,5)P2, and the P4M domain from Legionella SidM for PtdIns4P. Using accurately calibrated illumination, these indicated that only approximately 50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by approximately 100-fold dimmer intensities in mutants lacking Ca2+ permeable TRP channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t(1/2) approximately 12 s) was faster than PtdIns(4,5)P2 (t(1/2) approximately 40s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca2+ and PKC-dependent inhibition of PLC is critical for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle.

Choo, A., O'Keefe, L. V., Lee, C. S., Gregory, S. L., Shaukat, Z., Colella, A., Lee, K., Denton, D. and Richards, R. I. (2015). Tumor suppressor WWOX moderates the mitochondrial respiratory complex. Genes Chromosomes Cancer. PubMed ID: 26390919
Fragile site FRA16D exhibits DNA instability in cancer, resulting in diminished levels of protein from the WWOX gene that spans it. WWOX suppresses tumor growth by an undefined mechanism. WWOX participates in pathways involving aerobic metabolism and reactive oxygen species. WWOX comprises two WW domains as well as a short-chain dehydrogenase/reductase enzyme. This study describes an in vivo genetic analysis in Drosophila melanogaster to identify functional interactions between WWOX and metabolic pathways. Altered WWOX levels modulate variable cellular outgrowths caused by genetic deficiencies of components of the mitochondrial respiratory complexes. This modulation requires the enzyme active site of WWOX, and the defective respiratory complex-induced cellular outgrowths are mediated by reactive oxygen species, dependent upon the Akt pathway and sensitive to levels of autophagy and hypoxia-inducible factor. WWOX is known to contribute to homeostasis by regulating the balance between oxidative phosphorylation and glycolysis. Reduction of WWOX levels results in diminished ability to respond to metabolic perturbation of normal cell growth. Thus, the ability of WWOX to facilitate escape from mitochondrial damage-induced glycolysis (Warburg effect) is, therefore, a plausible mechanism for its tumor suppressor activity.

Khire, A., Vizuet, A. A., Davila, E. and Avidor-Reiss, T. (2015). Asterless reduction during spermiogenesis is regulated by Plk4 and is essential for zygote development in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 26480844
Centrosome reduction is the decrease in centrosomal components during spermatid differentiation (spermiogenesis). It is one of several dramatic subcellular reorganizations that lead to spermatozoa formation common to a wide range of animals. However, the mechanism underlying centrosome reduction is unknown and its functions are unclear. This study shows that in Drosophila melanogaster spermiogenesis, the quantity of centrosomal proteins is dramatically reduced; for example, Asterless (Asl) is reduced approximately 500-fold and is barely detected in spermatozoa. Asl reduction is regulated through a subset of its domains by the master regulator of centriole duplication Plk4 and by the ubiquitin ligase that targets Plk4 for degradation: Slimb. When Asl reduction is attenuated by Asl overexpression, plk4 mutations, Plk4 RNAi, or Slimb overexpression, Asl levels are higher in spermatozoa, resulting in embryos with reduced viability. Significantly, overexpressing Plk4 and Asl simultaneously, or combining plk4 and slimb mutations, balances their opposing effects on Asl reduction, restoring seemingly normal fertility. This suggests that increased Asl levels cause the observed reduced fertility and not other pleotropic effects. Attenuation of Asl reduction also causes delayed development and a failure to form astral microtubules in the zygote. Together, this study provides the first insight into a molecular mechanism that regulates centrosome reduction and the first direct evidence that centrosome reduction is essential for post-fertilization development.

Saturday, November 14th

Mattila, J., Havula, E., Suominen, E., Teesalu, M., Surakka, I., Hynynen, R., Kilpinen, H., Väänänen, J., Hovatta, I., Käkelä, R., Ripatti, S., Sandmann, T. and Hietakangas, V. (2015). Mondo-Mlx mediates organismal sugar sensing through the Gli-similar transcription factor Sugarbabe. Cell Rep [Epub ahead of print]. PubMed ID: 26440885
The ChREBP/Mondo-Mlx transcription factors are activated by sugars and are essential for sugar tolerance. They promote the conversion of sugars to lipids, but beyond this, their physiological roles are insufficiently understood. This study demonstrates that in an organism-wide setting in Drosophila, Mondo-Mlx controls the majority of sugar-regulated genes involved in nutrient digestion and transport as well as carbohydrate, amino acid, and lipid metabolism. Furthermore, human orthologs of the Mondo-Mlx targets display enrichment among gene variants associated with high circulating triglycerides. In addition to direct regulation of metabolic genes, Mondo-Mlx maintains metabolic homeostasis through downstream effectors, including the Activin ligand Dawdle and the Gli-similar transcription factor Sugarbabe. Sugarbabe controls a subset of Mondo-Mlx-dependent processes, including de novo lipogenesis and fatty acid desaturation. In sum, Mondo-Mlx is a master regulator of other sugar-responsive pathways essential for adaptation to a high-sugar diet.

Marada, S., Truong, A. and Ogden, S. K. (2015). The small GTPase Rap1 is a modulator of Hedgehog signaling. Dev Biol [Epub ahead of print]. PubMed ID: 26481064
During development, the evolutionarily conserved Hedgehog (Hh) morphogen provides instructional cues that influence cell fate, cell affinity and tissue morphogenesis. To do so, the Hh signaling cascade must coordinate its activity with other morphogenetic signals. This can occur through engagement of or response to effectors that do not typically function as core Hh pathway components. Given the ability of small G proteins of the Ras family to impact cell survival, differentiation, growth and adhesion, it was of interest to determine whether Hh and Ras signaling might intersect during development. Genetic modifier tests were performed in Drosophila to examine the ability of select Ras family members to influence Hh signal output, and Rap1 was identified as a positive modulator of Hh pathway activity. The results suggest that Rap1 is activated to its GTP-bound form in response to Hh ligand, and that the GTPase exchange factor C3G likely contributes to this activation. The Rap1 effector Canoe (Cno) also impacts Hh signal output, suggesting that a C3G-Rap1-Cno axis intersects the Hh pathway during tissue morphogenesis.

Deshpande, S.A., Yamada, R., Mak, C.M., Hunter, B., Soto Obando, A., Hoxha, S. and Ja, W.W. (2015). Acidic food pH increases palatability and consumption and extends Drosophila lifespan. J Nutr [Epub ahead of print]. PubMed ID: 26491123
Despite the prevalent use of Drosophila as a model in studies of nutrition, the effects of fundamental food properties, such as pH, on animal health and behavior are not well known. This study examined the effect of food pH on adult Drosophila lifespan, feeding behavior, and microbiota composition and tested the hypothesis that pH-mediated changes in palatability and total consumption are required for modulating longevity. The effect of buffered food (pH 5, 7, or 9) was measured on male gustatory responses (proboscis extension), total food intake, and male and female lifespan. The effect of food pH on germfree male lifespan was also assessed. Changes in fly-associated microbial composition as a result of food pH were determined by 16S ribosomal RNA gene sequencing. Male gustatory responses, total consumption, and male and female longevity were additionally measured in the taste-defective Pox neuro (Poxn) mutant and its transgenic rescue control. An acidic diet increases Drosophila gustatory responses (40-230%) and food intake (5-50%) and extends survival (10-160% longer median lifespan) compared with flies on either neutral or alkaline pH food. Alkaline food pH shifts the composition of fly-associated bacteria and results in greater lifespan extension (260% longer median survival) after microbes are eliminated compared with flies on an acidic (50%) or neutral (130%) diet. However, germfree flies live longer on an acidic diet (5-20% longer median lifespan) compared with those on either neutral or alkaline pH food. Gustatory responses, total consumption, and longevity are unaffected by food pH in Poxn mutant flies. Food pH can directly influence palatability and feeding behavior and affect parameters such as microbial growth to ultimately affect Drosophila lifespan. Fundamental food properties altered by dietary or drug interventions may therefore contribute to changes in animal physiology, metabolism, and survival.

Camus, M. F., Wolf, J. B., Morrow, E. H. and Dowling, D. K. (2015). Single nucleotides in the mtDNA sequence modify mitochondrial molecular function and are associated with sex-specific effects on fertility and aging. Curr Biol 25: 2717-2722. PubMed ID: 26455309
Mitochondria underpin energy conversion in eukaryotes. Their small genomes have been the subject of increasing attention, and there is evidence that mitochondrial genetic variation can affect evolutionary trajectories and shape the expression of life-history traits considered to be key human health indicators. However, it is not understood how genetic variation across a diminutive genome, which in most species harbors only about a dozen protein-coding genes, can exert broad-scale effects on the organismal phenotype. Such effects are particularly puzzling given that the mitochondrial genes involved are under strong evolutionary constraint and that mitochondrial gene expression is highly conserved across diverse taxa. This study used replicated genetic lines in the fruit fly, Drosophila melanogaster, each characterized by a distinct and naturally occurring mitochondrial haplotype placed alongside an isogenic nuclear background. Sequence variation within the mitochondrial DNA (mtDNA) affects both the copy number of mitochondrial genomes and patterns of gene expression across key mitochondrial protein-coding genes. In several cases, haplotype-mediated patterns of gene expression were gene-specific, even for genes from within the same transcriptional units. This invokes post-transcriptional processing of RNA in the regulation of mitochondrial genetic effects on organismal phenotypes. Notably, the haplotype-mediated effects on gene expression could be traced backward to the level of individual nucleotides and forward to sex-specific effects on fertility and longevity. This study thus elucidates how small-scale sequence changes in the mitochondrial genome can achieve broad-scale regulation of health-related phenotypes and even contribute to sex-related differences in longevity.

Friday, November 13th

Muhammad, K., et al. (2015). Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function. Nat Commun 6: 8362. PubMed ID: 26471740
Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. This study reports that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. It is concluded that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis.

Chen, Z., Chen, H. C. and Montell, C. (2015). TRP and Rhodopsin transport depends on dual XPORT ER chaperones encoded by an operon. Cell Rep 13: 573-584. PubMed ID: 26456832
TRP channels and G protein-coupled receptors (GPCRs) play critical roles in sensory reception. However, the identities of the chaperones that assist GPCRs in translocating from the endoplasmic reticulum (ER) are limited, and TRP ER chaperones are virtually unknown. The one exception for TRPs is Drosophila XPORT. This study shows that the xport locus is bicistronic and encodes unrelated transmembrane proteins, which enable the signaling proteins that initiate and culminate phototransduction, rhodopsin 1 (Rh1) and TRP, to traffic to the plasma membrane. XPORT-A (CG4468) and XPORT-B are ER proteins, and loss of either has a profound impact on TRP and Rh1 targeting to the light-sensing compartment of photoreceptor cells. XPORT-B complexed in vivo with the Drosophila homolog of the mammalian HSP70 protein, GRP78/BiP (Heat shock 70-kDa protein cognate 3), which, in turn, associated with Rh1. This work highlights a coordinated network of chaperones required for the biosynthesis of the TRP channel and rhodopsin in Drosophila photoreceptor cells.

Hicks, L., Liu, G., Ukken, F. P., Lu, S., Bollinger, K. E., O'Connor-Giles, K. and Gonsalvez, G. B. (2015). Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology. Biol Open [Epub ahead of print]. PubMed ID: 26459243
The mammalian Sorting Nexin 9 (Snx9) contains an Sh3 domain at its N-terminus and interacts with Dynamin and actin nucleation factors via this domain. Snx9 also contains a C-terminal BAR domain, known to sense and/or induce membrane curvature. The Snx9 family is encoded by a single gene in Drosophila called sh3px1. This report presents an initial characterization of sh3px1. Depletion of Sh3px1 from Drosophila Schneider 2 (S2) cells resulted in defective lamellipodia formation. A similar phenotype has been reported upon depletion of Scar, the actin nucleation factor implicated in forming lamellipodia. In addition, over-expression of Sh3px1 in S2 cells results in the formation of tubules as well as long protrusions. Formation of these structures required the C-terminal BAR domain as well as the adjacent Phox homology (PX) domain of Sh3px1. Furthermore, efficient protrusion formation by Sh3px1 required the actin nucleation factor Wasp. Tubules and protrusions were also generated upon over-expressing the mammalian orthologs Snx18 and Snx33 in S2 cells. By contrast, over-expressing Snx9 mostly induced long tubules.

Peng, Y., Lee, J., Rowland, K., Wen, Y., Hua, H., Carlson, N., Lavania, S., Parrish, J. Z. and Kim, M. D. (2015). Regulation of dendrite growth and maintenance by exocytosis. J Cell Sci [Epub ahead of print]. PubMed ID: 26483382
Dendrites lengthen by several orders of magnitude during neuronal development, but how membrane is allocated in dendrites to facilitate this growth remains unclear. This study reports that Ras opposite (Rop), the Drosophila ortholog of the key exocytosis regulator Munc18-1, is an essential factor mediating dendrite growth. Neurons with depleted Rop function exhibit reduced terminal dendrite outgrowth followed by primary dendrite degeneration, suggestive of differential requirements for exocytosis in the growth and maintenance of different dendritic compartments. Rop promotes dendrite growth together with the exocyst, an octameric protein complex involved in tethering vesicles to the plasma membrane, with Rop-exocyst complexes and exocytosis predominating in primary dendrites over terminal dendrites. By contrast, membrane-associated proteins readily diffuse from primary dendrites into terminals, but not in the reverse direction, suggesting that diffusion, rather than targeted exocytosis, supplies membranous material for terminal dendritic growth, revealing key differences in the distribution of materials to these expanding dendritic compartments.

Thursday, November 12th

Dhanyasi, N., Segal, D., Shimoni, E., Shinder, V., Shilo, B.Z., VijayRaghavan, K. and Schejter, E.D. (2015). Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles. J Cell Biol 211: 191-203. PubMed ID: 26459604
Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. This study used electron microscopy methods to study this key form of cell-cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. It was found that IFM myoblast-myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Cell adhesion was found to be necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell-cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process.

Loganathan, R., Lee, J. S., Wells, M. B., Grevengoed, E., Slattery, M. and Andrew, D. J. (2015). Ribbon regulates morphogenesis of the Drosophila embryonic salivary gland through transcriptional activation and repression. Dev Biol. PubMed ID: 26477561
Ribbon (Rib) controls cell shape/volume increases during elongation of the Drosophila salivary gland (SG), without effects on general SG cell attributes such as specification, proliferation and apoptosis and without compromising epithelial-specific morphological attributes. To identify the genes regulated by Rib, ChIP-seq analysis was performed in embryos driving expression of GFP-tagged Rib specifically in the SGs. Microarray analysis compared RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, 60 genomic sites bound by Rib were identified that were likely to regulate SG-specific gene expression. Several of the identified Rib targets were identified by qRT-pCR and/or in situ hybridization. The results indicate that Rib regulates cell growth and tissue shape via a diverse array of targets through both transcriptional activation and repression. Furthermore, the results suggest that autoregulation of rib expression may be a key component of the SG morphogenetic gene network.

Seong, K.H., Tsuda, M., Tsuda-Sakurai, K. and Aigaki, T. (2015). The plant homeodomain finger protein MESR4 is essential for embryonic development in Drosophila. Genesis [Epub ahead of print]. PubMed ID: 26467775
Misexpression Suppressor of Ras 4 (MESR4), a PHD finger protein with nine zinc-finger motifs has been implicated in various biological processes including the regulation of fat storage and innate immunity in Drosophila. However, the role of MESR4 in the context of development remains unclear. This study shows that MESR4 is a nuclear protein essential for embryonic development. Immunostaining of polytene chromosomes using anti-MESR4 antibody revealed that MESR4 binds to numerous bands along the chromosome arms. The most intense signal was detected at the 39E-F region, which is known to contain the histone gene cluster. P-element insertions in the MESR4 locus are homozygous lethal during embryogenesis with defects in ventral ectoderm formation and head encapsulation. In the mutant embryos, expression of Fasciclin 3 (Fas3), an EGFR signal target gene is greatly reduced, and the level of EGFR signal-dependent double phosphorylated ERK (dp-ERK) remains low. However, in the context of wing vein formation, genetic interaction experiments suggest that MESR4 is involved in the EGFR signaling as a negative regulator. These results suggest that MESR4 is a novel chromatin-binding protein required for proper expression of genes including those regulated by the EGFR signaling pathway during development.

Zheng, C., Diaz-Cuadros, M. and Chalfie, M. (2015). Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans. Proc Natl Acad Sci U S A 112: 13243-13248. PubMed ID: 26460008
Wnt proteins regulate axonal outgrowth along the anterior-posterior axis, but the intracellular mechanisms that modulate the strength of Wnt signaling in axon guidance are largely unknown. Using the Caenorhabditis elegans mechanosensory PLM neurons, this study found that posteriorly enriched LIN-44/Wnt acts as a repellent to promote anteriorly directed neurite outgrowth through the LIN-17/Frizzled (see Drosophila Frizzled) receptor, instead of controlling neuronal polarity as previously thought. Dishevelled proteins (see Drosophila Dishevelled) DSH-1 and MIG-5 redundantly mediate the repulsive activity of the Wnt signals to induce anterior outgrowth, whereas DSH-1 also provides feedback inhibition to attenuate the signaling to allow posterior outgrowth against the Wnt gradient. This inhibitory function of DSH-1, which requires its dishevelled, Egl-10, and pleckstrin (DEP) domain, acts by promoting LIN-17 phosphorylation and is antagonized by planar cell polarity signaling components Van Gogh (VANG-1; see Drosophila Van Gogh) and Prickle (PRKL-1; see Drosophila Prickle). These results suggest that Dsh proteins both respond to Wnt signals to shape neuronal projections and moderate its activity to fine-tune neuronal morphology.

Wednesday, November 11th

Parker, J. and Struhl, G. (2015). Scaling the Drosophila wing: TOR-dependent target gene access by the Hippo pathway transducer Yorkie. Journal-PLoS Biol 13: e1002274. PubMed ID: 26474042
How cells integrate distinct inputs to generate organs of the appropriate size and shape is largely unknown. The transcriptional coactivator Yorkie (Yki, a YES-Associated Protein, or YAP) acts downstream of patterning morphogens and other tissue-intrinsic signals to promote organ growth. Yki activity is regulated primarily by the Warts/Hippo (Wts/Hpo) pathway, which impedes nuclear access of Yki by a cytoplasmic tethering mechanism. This study shows that the TOR pathway regulates Yki by a separate and novel mechanism in the Drosophila wing. Instead of controlling Yki nuclear access, TOR signaling governs Yki action after it reaches the nucleus by allowing it to gain access to its target genes. When TOR activity is inhibited, Yki accumulates in the nucleus but is sequestered from its normal growth-promoting target genes. TOR also promotes wing growth by liberating Yki from nuclear seclusion, a parallel pathway that is proposed to contribute to the scaling of wing size with nutrient availability.

Srivastava, A. (2015). A novel link between FMR gene and the JNK pathway provides clues to possible role in malignant pleural mesothelioma. FEBS Open Bio 5: 705-711. PubMed ID: 26425438
Malignant pleural mesothelioma (MPM) is an aggressive form of thoracic cancer with poor prognosis. While some studies have identified the molecular alterations associated with MPM, little is known about their role in MPM. For example, fragile X mental retardation (FMR) gene is up-regulated in MPM but its role in MPM is unknown. Utilizing Drosophila genetics, this study investigated the possible role FMR may be playing in MPM. Evidence is provided that suggests that FMR may contribute to tumorigenesis by up-regulating a matrix metalloprotease (MMP) and by degrading the basement membrane (BM), both important for tumor metastasis. A novel link between FMR and the JNK pathway was demonstrated and it was suggested that the effects of FMR in MPM could in part be mediated by up-regulation of the JNK pathway.

Ramirez, J., Martinez, A., Lectez, B., Lee, S. Y., Franco, M., Barrio, R., Dittmar, G. and Mayor, U. (2015). Proteomic analysis of the ubiquitin landscape in the Drosophila embryonic nervous system and the adult photoreceptor cells. PLoS One 10: e0139083. PubMed ID: 26460970
Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Using an in vivo biotinylation strategy this study has isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, the proof-of-principle validation is reported of two lysine residues required for nSyb ubiquitination. These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described in this study complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.

Zhang, L., et al. (2015). Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature 527: 100-104. PubMed ID: 26479035
The development of life-threatening cancer metastases at distant organs requires disseminated tumour cells' adaptation to, and co-evolution with, the drastically different microenvironments of metastatic sites. Cancer cells of common origin manifest distinct gene expression patterns after metastasizing to different organs. It is unclear when and how disseminated tumour cells acquire the essential traits from the microenvironment of metastatic organs that prime their subsequent outgrowth. This study shows that both human and mouse tumour cells with normal expression of PTEN, an important tumour suppressor, lose PTEN expression after dissemination to the brain, but not to other organs. The PTEN level in PTEN-loss brain metastatic tumour cells is restored after leaving the brain microenvironment. This brain microenvironment-dependent, reversible PTEN messenger RNA and protein downregulation is epigenetically regulated by microRNAs from brain astrocytes. Mechanistically, astrocyte-derived exosomes mediate an intercellular transfer of PTEN-targeting microRNAs to metastatic tumour cells, while astrocyte-specific depletion of PTEN-targeting microRNAs or blockade of astrocyte exosome secretion rescues the PTEN loss and suppresses brain metastasis in vivo. These findings demonstrate a remarkable plasticity of PTEN expression in metastatic tumour cells in response to different organ microenvironments, underpinning an essential role of co-evolution between the metastatic cells and their microenvironment during the adaptive metastatic outgrowth.

Tuesday, November 10th

Pavot, P., Carbognin, E. and Martin, J. R. (2015). PKA and cAMP/CNG channels independently regulate the cholinergic Ca(2+)-response of Drosophila mushroom body neurons. eNeuro 2 [Epub ahead of print]. PubMed ID: 26464971
This work investigated the role of mushroom bodies (MBs) in olfactory learning and memory. Advantage was taken of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. Neuronal Ca(2+)-activity was imaged continuously, over a long time period, and the nicotine-evoked Ca(2+)-response was caracterized. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, it was first shown that the Ca(2+)-response is proportional to the levels of cAMP. Second, it was reveal that an acute change in cAMP levels is sufficient to trigger a Ca(2+)-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca(2+)-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. These results provide insights into the complex Ca(2+)-response in the MBs, leading to the conclusion that cAMP modulates the Ca(2+)-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels.

Roessingh, S., Wolfgang, W. and Stanewsky, R. (2015). Loss of Drosophila melanogaster TRPA1 function affects "siesta" behavior but not synchronization to temperature cycles. J Biol Rhythms [Epub ahead of print]. PubMed ID: 26459465
To maintain synchrony with the environment, circadian clocks use a wide range of cycling sensory cues that provide input to the clock (zeitgebers), including environmental temperature cycles (TCs). There is some knowledge about which clock neuronal groups are important for temperature synchronization, knowledge on the temperature receptors and their signaling pathways that feed temperature information to the (neuronal) clock is lacking. Since TRPA1 is a well-known thermosensor that functions in a range of temperature-related behaviors, and it is potentially expressed in clock neurons, this study set out to test the putative role of TRPA1 in temperature synchronization of the circadian clock. Flies lacking TRPA1 are still able to synchronize their behavioral activity to TCs comparable to wild-type flies, both in 16o C : 25o C and 20o C : 29o C TCs. In addition, it was found that flies lacking TRPA1 show higher activity levels during the middle of the warm phase of 20 o C : 29o C TCs, and it was show that this TRPA1-mediated repression of locomotor activity during the 'siesta' is caused by a lack of sleep. Based on these data, it is concluded that the TRPA1 channel is not required for temperature synchronization in this broad temperature range but instead is required to repress activity during the warm part of the day.

Kamiyama, D., McGorty, R., Kamiyama, R., Kim, M. D., Chiba, A. and Huang, B. (2015). Specification of dendritogenesis site in Drosophila aCC motoneuron by membrane enrichment of Pak1 through Dscam1. Dev Cell 35: 93-106. PubMed ID: 26460947
Precise positioning of dendritic branches is a critical step in the establishment of neuronal circuitry. However, there is limited knowledge on how environmental cues translate into dendrite initiation or branching at a specific position. Through a combination of mutation, RNAi, and imaging experiments, this study found that a Dscam-Dock-Pak1 hierarchical interaction defines the stereotypical dendrite growth site in the Drosophila aCC motoneuron. This interaction localizes the Cdc42 effector Pak1 to the plasma membrane at the dendrite initiation site before the activation of Cdc42. Ectopic expression of membrane-anchored Pak1 overrides this spatial specification of dendritogenesis, confirming its function in guiding Cdc42 signaling. It was further discovered that Dscam1 localization in aCC occurs through an inter-neuronal contact that involves Dscam1 in the partner MP1 neuron. These findings elucidate a mechanism by which Dscam1 controls neuronal morphogenesis through spatial regulation of Cdc42 signaling and, subsequently, cytoskeletal remodeling.

Fisher, Y. E., Silies, M. and Clandinin, T. R. (2015). Orientation selectivity sharpens motion detection in Drosophila.Neuron 88: 390-402. PubMed ID: 26456048
Detecting the orientation and movement of edges in a scene is critical to visually guided behaviors of many animals. What are the circuit algorithms that allow the brain to extract such behaviorally vital visual cues? Using in vivo two-photon calcium imaging in Drosophila, this study describes direction selective signals in the dendrites of T4 and T5 neurons, detectors of local motion. This circuit performs selective amplification of local light inputs, an observation that constrains motion detection models and confirms a core prediction of the Hassenstein-Reichardt correlator (HRC). These neurons are also orientation selective, responding strongly to static features that are orthogonal to their preferred axis of motion, a tuning property not predicted by the HRC. This coincident extraction of orientation and direction sharpens directional tuning through surround inhibition and reveals a striking parallel between visual processing in flies and vertebrate cortex, suggesting a universal strategy for motion processing.

Monday, November 9th

Eisman, R. C., Phelps, M. A. and Kaufman, T. (2015). An amino-terminal Polo kinase interaction motif acts in the regulation of centrosome formation and reveals a novel function for centrosomin (cnn) in Drosophila. Genetics 201: 685-706. PubMed ID: 26447129
The formation of the pericentriolar matrix (PCM) and a fully functional centrosome in syncytial Drosophila embryos requires the rapid transport of Cnn during initiation of the centrosome replication cycle. A Cnn and Polo kinase interaction is apparently required during embryogenesis and involves the exon 1A-initiating coding exon, suggesting a subset of Cnn splice variants is regulated by Polo kinase. During PCM formation exon 1A Cnn-Long Form proteins likely bind Polo kinase before phosphorylation by Polo for Cnn transport to the centrosome. Loss of either of these interactions in a portion of the total Cnn protein pool is sufficient to remove native Cnn from the pool, thereby altering the normal localization dynamics of Cnn to the PCM. Additionally, Cnn-Short Form proteins are required for polar body formation, a process known to require Polo kinase after the completion of meiosis. Exon 1A Cnn-LF and Cnn-SF proteins, in conjunction with Polo kinase, are required at the completion of meiosis and for the formation of functional centrosomes during early embryogenesis.

Christophorou, N., Rubin, T., Bonnet, I., Piolot, T., Arnaud, M. and Huynh, J. R. (2015). Microtubule-driven nuclear rotations promote meiotic chromosome dynamics. Nat Cell Biol 17: 1388-1400. PubMed ID: 26458247
At the onset of meiosis, each chromosome needs to find its homologue and pair to ensure proper segregation. In Drosophila, pairing occurs during the mitotic cycles preceding meiosis.This study shows that germ cell nuclei undergo marked movements during this developmental window. Microtubules and Dynein are driving nuclear rotations and are required for centromere pairing and clustering. It was further found that Klaroid (SUN) and Klarsicht (KASH) co-localize with centromeres at the nuclear envelope and are required for proper chromosome motions and pairing. Mud (NuMA in vertebrates) was identified as co-localizing with centromeres, Klarsicht and Klaroid. Mud is also required to maintain the integrity of the nuclear envelope and for the correct assembly of the synaptonemal complex. These findings reveal a mechanism for chromosome pairing in Drosophila, and indicate that microtubules, centrosomes and associated proteins play a crucial role in the dynamic organization of chromosomes inside the nucleus.

Drpic, D., Pereira, A. J., Barisic, M., Maresca, T. J. and Maiato, H. (2015). Polar ejection forces promote the conversion from lateral to end-on kinetochore-microtubule attachments on mono-oriented chromosomes. Cell Rep 13: 460-468. PubMed ID: 26456825
Chromosome bi-orientation occurs after conversion of initial lateral attachments between kinetochores and spindle microtubules into stable end-on attachments near the cell equator. After bi-orientation, chromosomes experience tension from spindle forces, which plays a key role in the stabilization of correct kinetochore-microtubule attachments. However, how end-on kinetochore-microtubule attachments are first stabilized in the absence of tension remains a key unanswered question. To address this, Drosophila S2 cells undergoing mitosis with unreplicated genomes (SMUGs) were generated. SMUGs retained single condensed chromatids that attached laterally to spindle microtubules. Over time, laterally attached kinetochores converted into end-on attachments and experienced intra-kinetochore stretch/structural deformation, and SMUGs eventually exited a delayed mitosis with mono-oriented chromosomes after satisfying the spindle-assembly checkpoint (SAC). Polar ejection forces (PEFs) generated by Chromokinesins promoted the conversion from lateral to end-on kinetochore-microtubule attachments that satisfied the SAC in SMUGs. Thus, PEFs convert lateral to stable end-on kinetochore-microtubule attachments, independently of chromosome bi-orientation.

Parker, D., Iyer, A., Shah, S., Moran, A., Hjelmeland, A., Basu, M. K., Liu, R. and Mitra, K. (2015). A novel mitochondrial pool of Cyclin E, regulated by Drp1, is linked to cell density dependent cell proliferation. J Cell Sci [Epub ahead of print]. PubMed ID: 26446260
The regulation and function of the crucial cell cycle regulator Cyclin E (CycE) remains elusive. Among other cyclins, CycE can be uniquely controlled by mitochondrial energetics, the exact mechanism being unclear. Using mammalian cells (in vitro) and Drosophila (in vivo) model systems in parallel this study shows that CycE can be directly regulated by mitochondria by its recruitment to the organelle. Active mitochondrial bioenergetics maintains a distinct mitochondrial pool of CycE (mtCycE) lacking a key phosphorylation required for its degradation. Loss of the mitochondrial fission protein Drp1 augments mitochondrial respiration and elevates the mtCycE-pool allowing CycE deregulation, cell cycle alterations and enrichment of stem cell markers. Such CycE deregulation after Drp1 loss attenuates cell proliferation in low cell density environments. However, in high cell density environments elevated MEK-ERK signaling in the absence of Drp1 releases mtCycE to support escape of contact inhibition and maintain aberrant cell proliferation. Such Drp1 driven regulation of CycE recruitment to mitochondria may be a mechanism to modulate CycE degradation during normal developmental processes as well as in tumorigenic events.

Sunday, November 8th

Yu, Y., Gu, J., Jin, Y., Luo, Y., Preall, J. B., Ma, J., Czech, B. and Hannon, G. J. (2015). Panoramix enforces piRNA-dependent cotranscriptional silencing. Science 350: 339-342. PubMed ID: 26472911
The Piwi-interacting RNA (piRNA) pathway is a small RNA-based innate immune system that defends germ cell genomes against transposons. In Drosophila ovaries, the nuclear Piwi protein is required for transcriptional silencing of transposons, though the precise mechanisms by which this occurs are unknown. This study show that the CG9754 protein is a component of Piwi complexes that functions downstream of Piwi and its binding partner, Asterix, in transcriptional silencing. Enforced tethering of CG9754 to nascent messenger RNA transcripts causes cotranscriptional silencing of the source locus and the deposition of repressive chromatin marks. CG9754 has been named "Panoramix," and it is proposed that this protein could act as an adaptor, scaffolding interactions between the piRNA pathway and the general silencing machinery that it recruits to enforce transcriptional repression.

Pek, J.W., Osman, I., Tay, M.L. and Zheng, R.T. (2015). Stable intronic sequence RNAs have possible regulatory roles in Drosophila melanogaster. J Cell Biol 211: 243-251. PubMed ID: 26504165
Stable intronic sequence RNAs (sisRNAs) have been found in Xenopus tropicalis, human cell lines, and Epstein-Barr virus; however, the biological significance of sisRNAs remains poorly understood. This study identified sisRNAs in Drosophila melanogaster by deep sequencing, reverse transcription polymerase chain reaction, and Northern blotting. A sisRNA (sisR-1) was characterized from the regena (rga) locus and was found to be processed from the precursor messenger RNA (pre-mRNA). A cis-natural antisense transcript (ASTR) from the rga locus was also documented that is highly expressed in early embryos. During embryogenesis, ASTR promotes robust rga pre-mRNA expression. Interestingly, sisR-1 represses ASTR, with consequential effects on rga pre-mRNA expression. These results suggest a model in which sisR-1 modulates its host gene expression by repressing ASTR during embryogenesis. The study proposes that sisR-1 belongs to a class of sisRNAs with probable regulatory activities in Drosophila.

Ryu, Y. H. and Macdonald, P. M. (2015). RNA sequences required for the noncoding function of oskar RNA also mediate regulation of Oskar protein expression by Bicoid Stability Factor. Dev Biol [Epub ahead of print]. PubMed ID: 26433064
The Drosophila oskar (osk) mRNA is unusual in having both coding and noncoding functions. As an mRNA, osk encodes a protein which is deployed specifically at the posterior of the oocyte. This spatially-restricted deployment relies on a program of mRNA localization and both repression and activation of translation, all dependent on regulatory elements located primarily in the 3' untranslated region (UTR) of the mRNA. The 3' UTR also mediates the noncoding function of osk, which is essential for progression through oogenesis. Mutations which most strongly disrupt the noncoding function are positioned in a short region (the C region) near the 3' end of the mRNA, in close proximity to elements required for activation of translation. This study show that Bicoid Stability Factor (BSF) binds specifically to the C region of the mRNA. Both knockdown of bsf and mutation of BSF binding sites in osk mRNA have the same consequences: Osk expression is largely eliminated late in oogenesis, with both mRNA localization and translation disrupted. Although the C region of the osk 3' UTR is required for the noncoding function, BSF binding does not appear to be essential for that function.

Germain, D. R., Li, L., Hildebrandt, M. R., Simmonds, A. J., Hughes, S. C. and Godbout, R. (2015). Loss of the Drosophila melanogaster DEAD box protein Ddx1 leads to reduced size and aberrant gametogenesis. Dev Biol [Epub ahead of print]. PubMed ID: 26433063
Mammalian DEAD box helicase DDX1 has been implicated in RNA trafficking, DNA double-strand break repair and RNA processing; however, little is known about its role during animal development. This study reports phenotypes associated with a null Ddx1 (Ddx1AX) mutation generated in Drosophila melanogaster. Ddx1 null flies are viable but significantly smaller than control and Ddx1 heterozygous flies. Female Ddx1 null flies have reduced fertility with egg chambers undergoing autophagy, whereas males are sterile due to disrupted spermatogenesis. Comparative RNA sequencing of control and Ddx1 null third instars identified several transcripts affected by Ddx1 inactivation. One of these, Sirup mRNA, was previously shown to be overexpressed under starvation conditions and implicated in mitochondrial function. This study demonstrates that Sirup is a direct binding target of Ddx1 and that Sirup mRNA is differentially spliced in the presence or absence of Ddx1. Combining Ddx1 null mutation with Sirup dsRNA-mediated knock-down causes epistatic lethality not observed in either single mutant. These data suggest a role for Drosophila Ddx1 in stress-induced regulation of splicing.

Saturday, November 7th

Rao, P.R., Lin, L., Huang, H., Guha, A., Roy, S. and Kornberg, T.B. (2015). Developmental compartments in the larval trachea of Drosophila. Elife [Epub ahead of print]. PubMed ID: 26491942
The Drosophila tracheal system is a branched tubular network that forms in the embryo by a post-mitotic program of morphogenesis. In third instar larvae (L3), cells constituting the second tracheal metamere (Tr2) reenter the cell cycle. Clonal analysis of L3 Tr2 reveals that dividing cells in the dorsal trunk, dorsal branch and transverse connective branches respect lineage restriction boundaries near branch junctions. These boundaries corresponded to domains of gene expression, for example where cells expressing Spalt, Delta and Serrate in the dorsal trunk meet vein-expressing cells in the dorsal branch or transverse connective. Notch signaling was activated to one side of these borders and was required for the identity, specializations and segregation of border cells. These findings suggest that Tr2 is comprised of developmental compartments and that developmental compartments are an organizational feature relevant to branched tubular networks.

Nikolova, L. S. and Metzstein, M. M. (2015). Intracellular lumen formation in Drosophila proceeds via a novel subcellular compartment. Development [Epub ahead of print]. PubMed ID: 26428009
To characterize the cellular mechanisms of subcellular tube formation, this study refined methods of high pressure freezing/freeze substitution to prepare Drosophila larvae for transmission electron microscopic (TEM) analysis. Using these methods, it was found that subcellular tracheal tube formation may proceed through a previously undescribed multimembrane intermediate composed of vesicles bound within a novel subcellular compartment. Correlative light/TEM procedures were developed to identify labeled cells in TEM-fixed larval samples. Using this technique, it was found that the vacuolar ATPase (V-ATPase) and the V-ATPase regulator Rabconnectin-3 are required for subcellular tube formation, probably in a step resolving the intermediate compartment into a mature lumen. In general, these methods should be applicable to analyzing the many cell biological problems which can be addressed using Drosophila larvae.

Diao, F., Mena, W., Shi, J., Park, D., Diao, F., Taghert, P., Ewer, J. and White, B. H. (2015). The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles. Genetics [Epub ahead of print]. PubMed ID: 26534952
In order to grow, insects must periodically shed their exoskeletons. This process, called ecdysis, is initiated by the endocrine release of Ecdysis Triggering Hormone (ETH) and has been extensively studied as a model for understanding the hormonal control of behavior. Understanding how ETH regulates ecdysis behavior, however, has been impeded by limited knowledge of the hormone's neuronal targets. An alternatively spliced gene encoding a G-protein coupled receptor (ETHR) that is activated by ETH has been identified, and several lines of evidence support a role in ecdysis for its A-isoform. The function of a second ETHR isoform (ETHRB) remains unknown. This study used the recently introduced 'Trojan exon' technique to simultaneously mutate the ETHR gene and gain genetic access to the neurons that express its two isoforms. ETHRA and ETHRB were shown to be expressed in largely distinct subsets of neurons, and ETHRA- , but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. However, both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval, ecdysis. Several functionally important subsets of ETHR-expressing neurons were found including one that co-expresses the peptide Leucokinin and regulates fluid balance to facilitate ecdysis at the pupal stage. The general strategy of using a receptor gene as an entry point for genetic and neuronal manipulations should be useful in establishing patterns of functional connectivity in other hormonally regulated networks.

Llorens, J. V., Metzendorf, C., Missirlis, F. and Lind, M. I. (2015). Mitochondrial iron supply is required for the developmental pulse of ecdysone biosynthesis that initiates metamorphosis in Drosophila melanogaster. J Biol Inorg Chem [Epub ahead of print]. PubMed ID: 26468126
Synthesis of ecdysone, the key hormone that signals the termination of larval growth and the initiation of metamorphosis in insects, is carried out in the prothoracic gland by an array of iron-containing cytochrome P450s, encoded by the halloween genes. This study shows that mutants in Drosophila mitoferrin (dmfrn), the gene encoding a mitochondrial carrier protein implicated in mitochondrial iron import, fail to grow and initiate metamorphosis under dietary iron depletion or when ferritin function is partially compromised. In mutant dmfrn larvae reared under iron replete conditions, the expression of halloween genes is increased and 20-hydroxyecdysone (20E), the active form of ecdysone, is synthesized. In contrast, addition of an iron chelator to the diet of mutant dmfrn larvae disrupts 20E synthesis. Dietary addition of 20E has little effect on the growth defects, but enables approximately one-third of the iron-deprived dmfrn larvae to successfully turn into pupae and, in a smaller percentage, into adults. This partial rescue is not observed with dietary supply of ecdysone's precursor 7-dehydrocholesterol, a precursor in the ecdysone biosynthetic pathway. The findings reported in this study support the notion that a physiological supply of mitochondrial iron for the synthesis of iron-sulfur clusters and heme is required in the prothoracic glands of insect larvae for steroidogenesis. Furthermore, mitochondrial iron is also essential for normal larval growth.

Friday, November 6th

Bai, L. and Sehgal, A. (2015). Anaplastic lymphoma kinase acts in the Drosophila mushroom body to negatively regulate sleep. PLoS Genet 11: e1005611. PubMed ID: 26536237
Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk), the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1) to regulate growth and associative learning in flies. This study shows that Alk mutants have increased sleep. Using a targeted RNAi screen the negative effects of Alk on sleep were located to the mushroom body, a structure important for both sleep and memory. Mutations in Nf1 were shown to produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes.

Vogt, K., Yarali, A. and Tanimoto, H. (2015). Reversing stimulus timing in visual conditioning leads to memories with opposite valence in Drosophila. PLoS One 10: e0139797. PubMed ID: 26430885
Animals need to associate different environmental stimuli with each other regardless of whether they temporally overlap or not. Drosophila melanogaster displays olfactory trace conditioning, where an odor is followed by electric shock reinforcement after a temporal gap, leading to conditioned odor avoidance. Reversing the stimulus timing in olfactory conditioning results in the reversal of memory valence such that an odor that follows shock is later on approached (i.e. relief conditioning). This study explored the effects of stimulus timing on memory in another sensory modality, using a visual conditioning paradigm. Flies were found to form visual memories of opposite valence depending on stimulus timing and can associate a visual stimulus with reinforcement despite being presented with a temporal gap. These results suggest that associative memories with non-overlapping stimuli and the effect of stimulus timing on memory valence are shared across sensory modalities.

Pan, L., et al. (2015). Heterochromatin remodeling by CDK12 contributes to learning in Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26508632
Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. This study reports that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors.

Pathak, T., Agrawal, T., Richhariya, S., Sadaf, S. and Hasan, G. (2015). Store-operated calcium entry through Orai is required for transcriptional maturation of the flight circuit in Drosophila. J Neurosci 35: 13784-13799. PubMed ID: 26446229
Store operated calcium entry (SOCE) is thought to primarily regulate calcium homeostasis in neurons. Subsequent to identification of Orai as the SOCE channel in nonexcitable cells, investigation of Orai function in neurons demonstrated a requirement for SOCE in Drosophila flight. By analysis of an Orai mutant and by controlled expression of a dominant-negative Drosophila Orai transgene, this study shows that Orai-mediated SOCE is required in dopaminergic interneurons of the flight circuit during pupal development. Expression of dominant-negative Orai in dopaminergic neurons of pupae abolished flight. The loss of Orai-mediated SOCE alters transcriptional regulation of dopaminergic neurons, leading to downregulation of the enzyme tyrosine hydroxylase, which is essential for dopamine synthesis, and the dopamine transporter, which is required for dopamine uptake after synaptic release. These studies suggest that modulation of SOCE could serve as a novel mechanism for restoring dopamine levels in dopaminergic neurons.

Thursday, November 5th

Liu, Z., Yang, C. P., Sugino, K., Fu, C. C., Liu, L. Y., Yao, X., Lee, L. P. and Lee, T. (2015). . Opposing intrinsic temporal gradients guide neural stem cell production of varied neuronal fates. Science 350: 317-320. PubMed ID: 26472907
Neural stem cells show age-dependent developmental potentials, as evidenced by their production of distinct neuron types at different developmental times. Drosophila neuroblasts produce long, stereotyped lineages of neurons. This study sought factors that could regulate neural temporal fate by RNA-sequencing lineage-specific neuroblasts at various developmental times. Two RNA-binding proteins, IGF-II mRNA-binding protein (Imp) and Syncrip (Syp), display opposing high-to-low and low-to-high temporal gradients with lineage-specific temporal dynamics. Imp and Syp promote early and late fates, respectively, in both a slowly progressing and a rapidly changing lineage. Imp and Syp control neuronal fates in the mushroom body lineages by regulating the temporal transcription factor Chinmo translation. Together, the opposing Imp/Syp gradients encode stem cell age, specifying multiple cell fates within a lineage.

Özel, M.N., Langen, M., Hassan, B.A. and Hiesinger, P.R. (2015). Filopodial dynamics and growth cone stabilization in Drosophila visual circuit development. Elife [Epub ahead of print]. PubMed ID: 26512889
Filopodial dynamics are thought to control growth cone guidance, but the types and roles of growth cone dynamics underlying neural circuit assembly in a living brain are largely unknown. To address this issue, this study developed long-term, continuous, fast and high-resolution imaging of growth cone dynamics from axon growth to synapse formation in cultured Drosophila brains. Using R7 photoreceptor neurons as a model it was shown that >90% of the growth cone filopodia exhibit fast, stochastic dynamics that persist despite ongoing stepwise layer formation. Correspondingly, R7 growth cones stabilize early and change their final position by passive dislocation. N-Cadherin controls both fast filopodial dynamics and growth cone stabilization. Surprisingly, loss of N-Cadherin causes no primary targeting defects, but destabilizes R7 growth cones to jump between correct and incorrect layers. Hence, growth cone dynamics can influence wiring specificity without a direct role in target recognition and implement simple rules during circuit assembly.

Lincoln, B.L., Alabsi, S.H., Frendo, N., Freund, R. and Keller, L.C. (2015). Drosophila neuronal injury follows a temporal sequence of cellular events leading to degeneration at the neuromuscular junction. J Exp Neurosci 9(Suppl 2): 1-9. PubMed ID: 26512206
Neurodegenerative diseases affect millions of people worldwide, and as the global population ages, there is a critical need to improve understanding of the molecular and cellular mechanisms that drive neurodegeneration. At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components. This study uses an in vivo motor neuron injury assay to acutely induce neurodegeneration in order to follow the temporal order of events that occur following injury in Drosophila melanogaster. It was found that sites of injury can be rapidly identified based on structural defects to the neuronal cytoskeleton that result in disrupted axonal transport. Additionally, the neuromuscular junction accumulates ubiquitinated proteins prior to the neurodegenerative events, occurring at 24 hours post injury. These data provide insights into the early molecular events that occur during axonal and neuromuscular degeneration in a genetically tractable model organism. Importantly, the mechanisms that mediate neurodegeneration in flies are conserved in humans. Thus, these studies have implications for the understanding of the cellular and molecular events that occur in humans and will facilitate the identification of biomedically relevant targets for future treatments.

Giles, A. C., Opperman, K. J., Rankin, C. H. and Grill, B. (2015). Developmental function of the PHR protein RPM-1 is required for learning in Caenorhabditis elegans. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26464359
The PAM/Highwire/RPM-1 (PHR) proteins are signaling hubs that function as important regulators of neural development. Loss of function in C. elegans E3 ubiquitin-protein ligase rpm-1 and Drosophila Highwire results in failed axon termination, inappropriate axon targeting, and abnormal synapse formation. Very mild abnormalities in behavior have been found in animals lacking PHR protein function. Therefore, it was hypothesized that large defects in behavior might only be detected in scenarios in which evoked, prolonged circuit function is required, or in which behavioral plasticity occurs. rpm-1 loss-of-function mutants had relatively mild abnormalities in exploratory locomotion, but exhibited large defects in evoked responses to harsh touch and learning associated with tap habituation. Rescue analysis indicated that RPM-1 function in the mechanosensory neurons affects habituation. Transgenic expression of RPM-1 in adult animals failed to rescue habituation defects, consistent with developmental defects in rpm-1 mutants resulting in impaired habituation. Genetic analysis showed that other regulators of neuronal development that function in the rpm-1 pathway (including glo-4, fsn-1, and dlk-1) also affected habituation. Overall, these findings suggest that developmental defects in rpm-1 mutants manifest most prominently in behaviors that require protracted or plastic circuit function, such as learning.

Wednesday, November 4th

Lim, Y.M., Yagi, Y. and Tsuda, L. (2015). Cellular defense and sensory cell survival require distinct functions of ebi in Drosophila. PLoS One 10: e0141457. PubMed: 26524764
The innate immune response and stress-induced apoptosis are well-established signaling pathways related to cellular defense. NF-κB and AP-1 are redox-sensitive transcription factors that play important roles in those pathways. This study shows that Ebi, a Drosophila homolog of the mammalian co-repressor molecule transducin β-like 1 (TBL1), variously regulates the expression of specific genes that are targets of redox-sensitive transcription factors. In response to different stimuli, Ebi activates gene expression to support the acute immune response in fat bodies, whereas Ebi represses genes that are involved in apoptosis in photoreceptor cells. Thus, Ebi seems to act as a regulatory switch for genes that are activated or repressed in response to different external stimuli. These results offer clear in vivo evidence that the Ebi-containing co-repressor complex acts in a distinct manner to regulate transcription that is required for modulating the output of various processes during Drosophila development.

Momiuchi, Y., Kumada, K., Kuraishi, T., Takagaki, T., Aigaki, T., Oshima, Y. and Kurata, S. (2015). Role of phylogenetically conserved co-chaperone protein Droj2/DNAJA3 in NF-kappaB signaling. J Biol Chem [Epub ahead of print]. PubMed ID: 26245905
The nuclear factor κ B (NF-κB) pathway is a phylogenetically conserved signaling pathway with a central role in inflammatory and immune responses. This study demonstrates that a co-chaperone protein, Droj2/DNAJA3, is involved in the activation of canonical NF-κB signaling in flies and in human cultured cells. Overexpression of Droj2 induced the expression of an antimicrobial peptide in Drosophila. Conversely, Droj2 knockdown resulted in reduced expression of antimicrobial peptides and higher susceptibility to Gram-negative bacterial infection in flies. Similarly, Toll-like receptor-stimulated inhibitor of κB (IκB) phosphorylation and NF-κB activation was suppressed by DNAJA3 knockdown in human embryonic kidney 293 cells. IκB kinase overexpression-induced NF-κB phosphorylation was also compromised in the DNAJA3-knockdown cells. This study reveals a novel conserved regulator of the NF-κB pathway acting at the level of IκB phosphorylation.

Kanoh, H., Tong, L.L., Kuraishi, T., Suda, Y., Momiuchi, Y., Shishido, F. and Kurata, S. (2015). Genome-wide RNAi screening implicates the E3 ubiquitin ligase Sherpa in mediating innate immune signaling by Toll in Drosophila adults. Sci Signal 8: ra107. PubMed ID: 26508789
The Drosophila Toll pathway plays important roles in innate immune responses against Gram-positive bacteria and fungi. To identify previously uncharacterized components of this pathway, this study performed comparative, ex vivo, genome-wide RNA interference screening. In four screens, the Toll adaptor protein dMyd88, the downstream kinase Pelle, or the nuclear factor κB (NF-κB) homolog Dif were overexpressed, or Cactus, the Drosophila homolog of mammalian inhibitor of NF-κB was knocked down. On the basis of these screens, the E3 ubiquitin ligase Sherpa was identified as being necessary for the activation of Toll signaling. A loss-of-function sherpa mutant fly exhibits compromised production of antimicrobial peptides and enhances susceptibility to infection by Gram-positive bacteria. In cultured cells, Sherpa mediated ubiquitylation of dMyd88 and Sherpa itself, and Sherpa and Drosophila SUMO (small ubiquitin-like modifier) are required for the proper membrane localization of an adaptor complex containing dMyd88. These findings highlight a role for Sherpa in Drosophila host defense and suggest the SUMOylation-mediated regulation of dMyd88 functions in Toll innate immune signaling. 

Kanoh, H., Kuraishi, T., Tong, L. L., Watanabe, R., Nagata, S. and Kurata, S. (2015). Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract. Biochem Biophys Res Commun 467: 400-406. PubMed ID: 26427875
Damage-associated molecular patterns (DAMPs), so-called "danger signals," play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. This study demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, an ex vivo genome-wide RNAi screening was performed in Drosophila cultured cells, and several signaling factors were identified that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that the larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.

Takahashi, D., Garcia, B. L. and Kanost, M. R. (2015). Initiating protease with modular domains interacts with beta-glucan recognition protein to trigger innate immune response in insects. Proc Natl Acad Sci U S A. PubMed ID: 26504233
The autoactivation of an initiating serine protease upon binding of pattern recognition proteins to pathogen surfaces is a crucial step in eliciting insect immune responses such as the activation of Toll and prophenoloxidase pathways. However, the molecular mechanisms responsible for autoactivation of the initiating protease remains poorly understood. This study investigated the molecular basis for the autoactivation of hemolymph protease 14 (HP14), an initiating protease in hemolymph of Manduca sexta, upon the binding of beta-1,3-glucan by its recognition protein, βGRP2. Biochemical analysis using HP14 zymogen (proHP14), βGRP2, and the recombinant proteins as truncated forms showed that the amino-terminal modular low-density lipoprotein receptor class A (LA) domains within HP14 are required for proHP14 autoactivation that is stimulated by its interaction with βGRP2. Consistent with this result, recombinant LA domains inhibit the activation of proHP14 and prophenoloxidase, likely by competing with the interaction between βGRP2 and LA domains within proHP14. LA domains directly interact with βGRP2 in a calcium-dependent manner, and high-affinity interaction requires the C-terminal glucanase-like domain of βGRP2. Importantly, the affinity of LA domains for βGRP2 increases nearly 100-fold in the presence of beta-1,3-glucan. Taken together, these results present the first experimental evidence LA domains of an insect modular protease and glucanase-like domains of a βGRP mediate their interaction, and that this binding is essential for the protease autoactivation. Thus, this study provides important insight into the molecular basis underlying the initiation of protease cascade in insect immune responses.

Wang, Y. H., Hu, Y., Xing, L. S., Jiang, H., Hu, S. N., Raikhel, A. S. and Zou, Z. (2015). A critical role for CLSP2 in the modulation of antifungal immune response in mosquitoes. PLoS Pathog 11: e1004931. PubMed ID: 26057557
Entomopathogenic fungi represent a promising class of bio-insecticides for mosquito control. Thus, detailed knowledge of the molecular mechanisms governing anti-fungal immune response in mosquitoes is essential. This study shows that CLSP2, a serine protease with lectin domains, is a modulator of immune responses during anti-fungal infection in the mosquito Aedes aegypti. With a fungal infection, the expression of the CLSP2 gene is elevated. CLSP2 is cleaved upon challenge with Beauveria bassiana conidia, and the liberated CLSP2 C-terminal galactose-type C-type lectin (CTL)-type domain binds to fungal cell components and B. bassiana conidia. Furthermore, CLPS2 RNA interference silencing significantly increases the resistance to the fungal challenge. RNA-sequencing transcriptome analysis showed that the majority of immune genes were highly upregulated in the CLSP2-depleted mosquitoes infected with the fungus. The up-regulated immune gene cohorts belong to melanization and Toll pathways, but not to the IMD or JAK-STAT. The unique interaction of CLSP2 with Cactus suggests that it contributes in the control of AMP gene activation. Moreover, the abolishment of activation of AMPs, brought by iCLSP2 by the double knockdown of CLSP2 and Rel1, indicates that Rel1 mediates the action of CLSP2 on these immune genes. A thioester-containing protein (TEP22), a member of alpha2-macroglobulin family, has been implicated in the CLSP2-modulated mosquito antifungal defense. This study has contributed to a greater understanding of immune-modulating mechanisms in mosquitoes.

Tuesday, November 3rd

Garbe, D.S., Bollinger, W.L., Vigderman, A., Masek, P., Gertowski, J., Sehgal, A. and Keene, A.C. (2015). Context-specific comparison of sleep acquisition systems in Drosophila. Biol Open [Epub ahead of print]. PubMed ID: 26519516
Sleep is conserved across phyla and can be measured through electrophysiological or behavioral characteristics. The fruit fly, Drosophila melanogaster, provides an excellent model for investigating the genetic and neural mechanisms that regulate sleep. Multiple systems exist for measuring fly activity, including video analysis and single-beam (SB) or multi-beam (MB) infrared (IR)-based monitoring. This study compared multiple sleep parameters of individual flies using a custom-built video-based acquisition system, and commercially available SB- or MB-IR acquisition systems. It was found that all three monitoring systems appear sufficiently sensitive to detect changes in sleep duration associated with diet, age, and mating status. It was also demonstrated that MB-IR detection appears more sensitive than the SB-IR for detecting baseline nuances in sleep architecture, while architectural changes associated with varying life-history and environment are generally detected across all acquisition types. Finally, video recording of flies in an arena allows measurement of the effect of ambient environment on sleep. These experiments demonstrate a robust effect of arena shape and size as well as light levels on sleep duration and architecture, and highlighting the versatility of tracking-based sleep acquisition. These findings provide insight into the context-specific basis for choosing between Drosophila sleep acquisition systems, describe a novel cost-effective system for video tracking, and characterize sleep analysis using the MB-IR sleep analysis. Further, the study also describes a modified dark-place preference sleep assay using video tracking, confirming that flies prefer to sleep in dark locations.

Medina, I., Casal, J. and Fabre, C.C. (2015). Do circadian genes and ambient temperature affect substrate-borne signalling during Drosophila courtship? Biol Open [Epub ahead of print]. PubMed ID: 26519517
Courtship vibratory signals can be air-borne or substrate-borne. They convey distinct and species-specific information from one individual to its prospective partner. This study focuses on the substrate-borne vibratory signals generated by the abdominal quivers of the Drosophila male during courtship; these vibrations travel through the ground towards courted females and coincide with female immobility. It is not known which physical parameters of the vibrations encode the information that is received by the females and induces them to pause. The intervals between each vibratory pulse were examined, a feature that was reported to carry information for animal communication. However, evidence of periodic variations in the lengths of these intervals could not be found, as has been reported for fly acoustical signals. Because it has been suggested that the genes involved in the circadian clock may also regulate shorter rhythms, effects of period on the interval lengths were determined. Males that were mutant for the period gene produce vibrations with significantly altered interpulse intervals; also, treating wild type males with constant light results in similar alterations to the interpulse intervals. These results suggest that both the clock and light/dark cycles have input into the interpulse intervals of these vibrations. By altering the interpulse intervals by other means, it was found that ambient temperature also has a strong effect. However, behavioural analysis suggests that only extreme ambient temperatures can affect the strong correlation between female immobility and substrate-borne vibrations.

Lin, C. C., Prokop-Prigge, K. A., Preti, G. and Potter, C. J. (2015). Food odors trigger males to deposit a pheromone that guides aggregation and female oviposition decisions. Elife 4. PubMed ID: 26422512
Animals use olfactory cues for navigating complex environments. Food odors in particular provide crucial information regarding potential foraging sites. Many behaviors occur at food sites, yet how food odors regulate such behaviors at these sites is unclear. Using Drosophila melanogaster as an animal model, this study found that males deposit the pheromone 9-tricosene upon stimulation with the food-odor apple cider vinegar. This pheromone acts as a potent aggregation pheromone and as an oviposition guidance cue for females. Genetic, molecular, electrophysiological, and behavioral approaches were used to show that 9-tricosene activates antennal basiconic Or7a receptors, a receptor activated by many alcohols and aldehydes such as the green leaf volatile E2-hexenal. Loss of Or7a+ neurons or the Or7a receptor abolishes aggregation behavior and oviposition site-selection towards 9-tricosene and E2-hexenal. 9-Tricosene thus functions via Or7a to link food-odor perception with aggregation and egg-laying decisions.
Sellami, A. and Veenstra, J. A. (2015). SIFamide acts on fruitless neurons to modulate sexual behavior in Drosophila melanogaster. Peptides [Epub ahead of print]. PubMed ID: 26469541
The Drosophila gene fruitless expresses male and female specific transcription factors which are responsible for the generation of male specific neuronal circuitry for courtship behavior. Mutations in this gene may lead to bisexual behavior in males. Bisexual behavior in males also occurs in the absence of the neuropeptide SIFamide. SIFamide neurons do not express fruitless. However, when fruitless neurons are made to express RNAi specific for the SIFamide receptor, male flies engage in bisexual behavior, showing that SIFamide acts on fruitless neurons. If neurons expressing a SIFaR-gal4 transgene are killed by the apoptotic protein Reaper or when these neurons express SIFamide receptor RNAi, males also show male-male courtship behavior. This transgene was used to localize neurons that express the SIFamide receptor. Such neurons are ubiquitously present in the central nervous, and two neurons were also found in the uterus that project into the central nervous system.

Monday, November 2nd

Jia, M., Shan, Z., Yang, Y., Liu, C., Li, J, Luo, Z.G., Zhang, M., Cai, Y., Wen, W. and Wang, W. (2015). The structural basis of Miranda-mediated Staufen localization during Drosophila neuroblast asymmetric division. Nat Commun 6: 8381. PubMed ID: 26423004
During the asymmetric division of Drosophila neuroblasts (NBs), the scaffold Miranda (Mira) coordinates the subcellular distribution of cell-fate determinants including Staufen (Stau) and segregates them into the ganglion mother cells (GMCs). This study shows that the fifth double-stranded RNA (dsRNA)-binding domain (dsRBD5) of Stau is necessary and sufficient for binding to a coiled-coil region of Mira cargo-binding domain (CBD). The crystal structure of Mira514-595/Stau dsRBD5 complex illustrates that Mira forms an elongated parallel coiled-coil dimer, and two dsRBD5 symmetrically bind to the Mira dimer through their exposed β-sheet faces, revealing a previously unrecognized protein interaction mode for dsRBDs. It was further demonstrated that the Mira-Stau dsRBD5 interaction is responsible for the asymmetric localization of Stau during Drosophila NB asymmetric divisions. Finally, it was found that the CBD-mediated dimer assembly is likely a common requirement for Mira to recognize and translocate other cargos including brain tumour (Brat).

Geyer, A., Koltsaki, I., Hessinger, C., Renner, S. and Rogulja-Ortmann, A. (2015). Impact of Ultrabithorax alternative splicing on Drosophila embryonic nervous system development. Mech Dev [Epub ahead of print]. PubMed ID: 26299253
Hox genes control divergent segment identities along the anteroposterior body axis of bilateral animals by regulating a large number of processes in a cell context-specific manner. How Hox proteins achieve this functional diversity is a long-standing question in developmental biology. This study investigated the role of alternative splicing in functional specificity of the Drosophila Hox gene Ultrabithorax (Ubx). Focus was placed specifically on the embryonic central nervous system (CNS), and a description is provided of temporal expression patterns of three major Ubx isoforms during development of this tissue. These analyses imply distinct functions for individual isoforms in different stages of neural development. The set of Ubx isoforms expressed in two isoform-specific Ubx mutant strains was analyzed along with an analysis of the effects of splicing defects on regional neural stem cell (neuroblast) identity. These findings support the notion of specific isoforms having different effects in providing individual neuroblasts with positional identity along the anteroposterior body axis, as well as being involved in regulation of progeny cell fate.

Brown, H. E., Reichert, M. C. and Evans, T. A. (2015). Slit binding via the Ig1 domain is essential for midline repulsion by Drosophila Robo1 but dispensable for receptor expression, localization, and regulation in vivo. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26362767
This study examined the in vivo functional importance of the Ig1 domain of the Drosophila Roundabout1 receptor, which controls midline crossing of axons in response to Slit produced by the embryonic midline. Deleting Ig1 from Robo1 disrupts Slit binding in cultured Drosophila cells, and that a Robo1 variant lacking Ig1 (Robo1Ig1) is unable to promote ectopic midline repulsion in gain of function studies in the Drosophila embryonic CNS. The Ig1 domain is not required for proper expression, axonal localization, or Commissureless (Comm)-dependent regulation of Robo1 in vivo, and a genetic rescue assay was used to show that Robo1Ig1 is unable to substitute for full-length Robo1 to properly regulate midline crossing of axons. These results establish a direct link between in vitro biochemical studies of Slit-Robo interactions and in vivo genetic studies of Slit-Robo signaling during midline axon guidance, and distinguish Slit-dependent from Slit-independent aspects of Robo1 expression, regulation, and activity during embryonic development.

Kumar, R., Chotaliya, M., Vuppala, S., Auradkar, A., Palasamudrum, K. and Joshi, R. (2015). Role of Homothorax in region specific regulation of Deformed in embryonic neuroblasts. Mech Dev [Epub ahead of print]. PubMed ID: 26409112
The expression and regulation of Hox genes in developing central nervous system (CNS) lack important details like specific cell types where Hox genes are expressed and the transcriptional regulatory players involved in these cells. This study has investigated the expression and regulation of Drosophila Hox gene Deformed (Dfd) in specific cell types of embryonic CNS. Using Dfd neural autoregulatory enhancer, it was found that Dfd autoregulates itself in cells of mandibular neuromere. The role was investigated of a Hox cofactor Homothorax (Hth) for its role in regulating Dfd expression in CNS. Hth was found to exhibit a region specific role in controlling the expression of Dfd, but has no direct role in mandibular Dfd neural autoregulatory circuit. These results also suggest that homeodomain of Hth is not required for regulating Dfd expression in embryonic CNS.

Sunday, November 1st

Zhang, Z. and Presgraves, D.C. (2015). Drosophila X-linked genes have lower translation rates than autosomal genes. Mol Biol Evol [Epub ahead of print]. PubMed ID: 26486873
In Drosophila, X-linked and autosomal genes achieve comparable expression at the mRNA level. Whether comparable X-autosome gene expression is realized at the translational and, ultimately, the protein levels is however unknown. Previous studies suggest the possibility of higher translation rates for X-linked genes owing to stronger usage of preferred codons. This study uses public ribosome profiling data from Drosophila melanogaster to infer translation rates on the X chromosome versus the autosomes. It was found that X-linked genes have consistently lower ribosome densities than autosomal genes in S2 cells, early embryos, eggs, and mature oocytes. Surprisingly, the lower ribosome densities of X-linked genes are not consistent with faster translation elongation but instead imply slower translation initiation. In particular, X-linked genes have sequence features known to slow translation initiation such as stronger mRNA structure near start codons and longer 5' UTRs. Comparison to outgroup species suggests that stronger mRNA structure is an evolved feature of Drosophila X chromosomes. Finally, it was found that the magnitude of the X-autosome difference in ribosome densities is smaller for genes encoding members of protein complexes, suggesting that stoichiometry constrains the evolution of translation rates. In sum, these analyses suggest that Drosophila X-linked genes have evolved lower translation rates than autosomal genes despite stronger usage of preferred codons.

Calvo-Martín, J.M., Librado, P., Aguadé, M., Papaceit, M. and Segarra, C. (2015). Adaptive selection and coevolution at the proteins of the Polycomb repressive complexes in Drosophila. Heredity (Edinb) [Epub ahead of print]. PubMed ID: 26486609
Polycomb group (PcG) proteins are important epigenetic regulatory proteins that modulate the chromatin state through posttranslational histone modifications. These interacting proteins form multimeric complexes that repress gene expression. Thus, PcG proteins are expected to evolve coordinately, which might be reflected in their phylogenetic trees by concordant episodes of positive selection and by a correlation in evolutionary rates. In order to detect these signals of coevolution, this study analyzed the molecular evolution of 17 genes encoding the subunits of five Polycomb repressive complexes in the Drosophila genus. The observed distribution of divergence differs substantially among and along proteins. Indeed, CAF1 is uniformly conserved, whereas only the established protein domains are conserved in other proteins, such as PHO, PHOL, PSC, PH-P and ASX. Moreover, regions with a low divergence not yet described as protein domains are present, for instance, in SFMBT and SU(Z)12. Maximum likelihood methods indicate an acceleration in the nonsynonymous substitution rate at the lineage ancestral to the obscura group species in most genes encoding subunits of the Pcl-PRC2 complex and in genes Sfmbt, Psc and Kdm2. These methods also allow inferring the action of positive selection in this lineage at genes E(z) and Sfmbt. Finally, the protein interaction network predicted from the complete proteomes of 12 Drosophila species using a coevolutionary approach shows two tight PcG clusters. These clusters include well-established binary interactions among PcG proteins as well as new putative interactions.

Vedanayagam, J. P. and Garrigan, D. (2015). The effects of natural selection across molecular pathways in Drosophila melanogaster. BMC Evol Biol 15: 203. PubMed ID: 26391223
Whole-genome RNA interference post-transcriptional silencing (RNAi) is a widely used method for studying the phenotypic effects of knocking down individual genes. This study used a population genomic approach to characterize the rate of evolution for proteins affecting 26 RNAi knockdown phenotypes in Drosophila melanogaster. Only two of the 26 RNAi knockdown phenotypes are enriched for rapidly evolving proteins: innate immunity and regulation of Hedgehog signaling. Among all genes associated with an RNAi knockdown phenotype, examples were noted in which the adaptively evolving proteins play a well-defined role in a given molecular pathway. However, most adaptively evolving proteins are found to perform more general cellular functions. When RNAi phenotypes are grouped into categories according to cellular function, it was found that genes involved in the greatest number of phenotypic categories are also significantly more likely to have a history of rapid protein evolution. Genes that have been demonstrated to have a measurable effect on multiple molecular phenotypes show higher rates of protein evolution than genes having an effect on a single category of phenotype. Defining pleiotropy in this way yields very different results than previous studies that define pleiotropy by the number of physical interactions, which show highly connected proteins tend to evolve more slowly than lowly connected proteins. It is suggested that a high degree of pleiotropy may increase the likelihood of compensatory substitution, consistent with modern theoretical work on adaptation.

Yee, W. K., Rogell, B., Lemos, B. and Dowling, D. K. (2015). Intergenomic interactions between mitochondrial and Y-linked genes shape male mating patterns and fertility in Drosophila melanogaster. Evolution [Epub ahead of print]. PubMed ID: 26419212
Under maternal inheritance, mitochondrial genomes are prone to accumulate mutations that exhibit male-biased effects. Such mutations should, however, place selection on the nuclear genome for modifier adaptations that mitigate mitochondrial-incurred male harm. One gene region that might harbor such modifiers is the Y-chromosome, given the abundance of Y-linked variation for male fertility, and because Y-linked modifiers would not exert antagonistic effects in females because they would be found only in males. Recent studies in Drosophila revealed a set of nuclear genes whose expression is sensitive to allelic variation among mtDNA- and Y-haplotypes, suggesting these genes might be entwined in evolutionary conflict between mtDNA and Y. This study tested whether genetic variation across mtDNA and Y haplotypes, sourced from three disjunct populations, interacts to affect male mating patterns and fertility across 10 days of early life in D. melanogaster. Whether coevolved mito-Y combinations outperform their evolutionarily novel counterparts, as predicted if the interacting Y-linked variance is comprised of modifier adaptations, was also tested. Although no evidence was found that coevolved mito-Y combinations outperformed their novel counterparts, interactions between mtDNA and Y-chromosomes affected male mating patterns. These interactions were dependent on male age; thus male reproductive success was shaped by G x G x E interactions.

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