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Tuesday, December 31st, 2019 - Signaling

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Link, N., Chung, H., Jolly, A., Withers, M., Tepe, B., Arenkiel, B. R., Shah, P. S., Krogan, N. J., Aydin, H., Geckinli, B. B., Tos, T., Isikay, S., Tuysuz, B., Mochida, G. H., Thomas, A. X., Clark, R. D., Mirzaa, G. M., Lupski, J. R. and Bellen, H. J. (2019). Mutations in ANKLE2, a ZIKA virus target, disrupt an asymmetric cell division pathway in Drosophila neuroblasts to cause microcephaly. Dev Cell. PubMed ID: 31735666
Summary:
The apical Par complex, which contains atypical protein kinase C (aPKC), Bazooka (Par-3), and Par-6, is required for establishing polarity during asymmetric division of neuroblasts in Drosophila, and its activity depends on L(2)gl. This study shows that loss of Ankle2, a protein associated with microcephaly in humans and known to interact with Zika protein NS4A, reduces brain volume in flies and impacts the function of the Par complex. Reducing Ankle2 levels disrupts endoplasmic reticulum (ER) and nuclear envelope morphology, releasing the kinase -VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par-complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen or l(2)gl suppresses Ankle2 mutant phenotypes and restores viability and brain size. Human mutational studies implicate the above-mentioned genes in microcephaly and motor neuron disease. It is suggested that NS4A, ANKLE2, VRK1, and LLGL1 define a pathway impinging on asymmetric determinants of neural stem cell division.
Sarpal, R., Yan, V., Kazakova, L., Sheppard, L., Yu, J. C., Fernandez-Gonzalez, R. and Tepass, U. (2019). Role of alpha-Catenin and its mechanosensing properties in regulating Hippo/YAP-dependent tissue growth. PLoS Genet 15(11): e1008454. PubMed ID: 31697683
Summary:
alpha-catenin is a key protein of adherens junctions (AJs) with mechanosensory properties. It also acts as a tumor suppressor that limits tissue growth. This study analyzed the function of Drosophila alpha-Catenin (alpha-Cat) in growth regulation of the wing epithelium. Different alpha-Cat levels led to a differential activation of Hippo/Yorkie or JNK signaling causing tissue overgrowth or degeneration, respectively. alpha-Cat can modulate Yorkie-dependent tissue growth through recruitment of Ajuba, a negative regulator of Hippo signaling to AJs but also through a mechanism independent of Ajuba recruitment to AJs. Both mechanosensory regions of alpha-Cat, the M region and the actin-binding domain (ABD), contribute to growth regulation. Whereas M is dispensable for alpha-Cat function in the wing, individual M domains (M1, M2, M3) have opposing effects on growth regulation. In particular, M1 limits Ajuba recruitment. Loss of M1 causes Ajuba hyper-recruitment to AJs, promoting tissue-tension independent overgrowth. Although M1 binds Vinculin, Vinculin is not responsible for this effect. Moreover, disruption of mechanosensing of the alpha-Cat ABD affects tissue growth, with enhanced actin interactions stabilizing junctions and leading to tissue overgrowth. Together, these findings indicate that alpha-Cat acts through multiple mechanisms to control tissue growth, including regulation of AJ stability, mechanosensitive Ajuba recruitment, and dynamic direct F-actin interactions.
Park, S. H., Lee, C. W. and Choe, K. M. (2019). Interplay between integrins and PI4P5K Sktl is crucial for cell polarization and reepithelialisation during Drosophila wound healing. Sci Rep. 9(1):16331. PubMed ID: 31704968
Summary:
Phosphatidylinositol(4,5)-bisphosphate [PI(4,5)P2] regulates cell adhesion and actin dynamics during cell migration. PI(4,5)P2 binds various components of the cell adhesion machinery, but how these processes affect migration of the epithelial cell sheet is not well understood. This study shows that PI(4,5)P2 and Sktl, the kinase that converts PI4P to PI(4,5)P2, are both localized to the rear side of cells during wound healing of the Drosophila larval epidermis. The Sktl localization requires JNK pathway activation and integrins, but not PVR. The sktl knockdown epidermis displays strong defects in wound closure, reminiscent of the JNK-depleted epidermis, and shows severe disruption of cell polarity, as determined by myosin II localization. Sktl and betaPS integrin colocalize at the rear side of cells forming the trailing edge during wound healing and the two are inter-dependent in that the absence of one severely disrupts the rear localization of the other. These results strongly suggest that the JNK pathway regulates the rear localization of Sktl and integrins and the interplay between Sktl and integrins sets up cell polarity, which is crucial for reepithelialisation during wound healing.
Pandey, A., Harvey, B. M., Lopez, M. F., Ito, A., Haltiwanger, R. S. and Jafar-Nejad, H. (2019). Glycosylation of specific Notch EGF repeats by O-Fut1 and Fringe regulates Notch signaling in Drosophila. Cell Rep 29(7): 2054-2066. PubMed ID: 31722217
Summary:
Fringe glycosyltransferases differentially modulate the binding of Notch receptors to Delta/DLL versus Serrate/Jagged ligands by adding GlcNAc to O-linked fucose on Notch epidermal growth factor-like (EGF) repeats. Although Notch has 22 O-fucosylation sites, the biologically relevant sites affecting Notch activity during animal development in vivo in the presence or absence of Fringe are not known. Using a variety of assays, this study found important roles in Drosophila Notch signaling for GlcNAc-fucose-O glycans on three sites: EGF8, EGF9, and EGF12. O-Fucose monosaccharide on EGF12 (in the absence of Fringe) is essential for Delta-mediated lateral inhibition in embryos. However, wing vein development depends on the addition of GlcNAc to EGF8 and EGF12 by Fringe, with a minor contribution from EGF9. Fringe modifications of EGF8 and EGF12 together prevent Notch from cis-inhibiting Serrate, thereby promoting normal wing margin formation. This work shows the combinatorial and context-dependent roles of GlcNAc-fucose-O glycans on these sites in Drosophila Notch-ligand interactions.
Sidor, C., Borreguero-Munoz, N., Fletcher, G. C., Elbediwy, A., Guillermin, O. and Thompson, B. J. (2019). Mask family proteins ANKHD1 and ANKRD17 regulate YAP nuclear import and stability. Elife 8. PubMed ID: 31661072
Summary:
Mask family proteins were discovered in Drosophila to promote the activity of the transcriptional coactivator Yorkie (Yki), the sole fly homolog of mammalian YAP (YAP1) and TAZ (WWTR1). The molecular function of Mask, or its mammalian homologs Mask1 (ANKHD1) and Mask2 (ANKRD17), remains unclear. Mask family proteins contain two ankyrin repeat domains that bind Yki/YAP as well as a conserved nuclear localisation sequence (NLS) and nuclear export sequence (NES), suggesting a role in nucleo-cytoplasmic transport. This study shows that Mask acts to promote nuclear import of Yki, and that addition of an ectopic NLS to Yki is sufficient to bypass the requirement for Mask in Yki-driven tissue growth. Mammalian Mask1/2 proteins also promote nuclear import of YAP, as well as stabilising YAP and driving formation of liquid droplets. Mask1/2 and YAP normally colocalise in a granular fashion in both nucleus and cytoplasm, and are co-regulated during mechanotransduction.
Trautenberg, L. C., Prince, E., Maas, C., Beier, N., Honold, F., Grzybek, M. and Brankatschk, M. (2019). Selective phosphorylation of Akt/Protein-kinase B isoforms in response to dietary cues. Front Cell Dev Biol 7: 206. PubMed ID: 31649929
Summary:
A calorie-rich diet is one reason for the continuous spread of metabolic syndromes in western societies. Smart food design is one powerful tool to prevent metabolic stress, and the search for suitable bioactive additives is a continuous task. The nutrient-sensing insulin pathway is an evolutionary conserved mechanism that plays an important role in metabolism, growth and development. Recently, lipid cues capable to stimulate insulin signaling were identified. However, the mechanistic base of their activity remains obscure to date. This study shows that specific Akt/Protein-kinase B isoforms are responsive to different calorie-rich diets, and potentiate the activity of the cellular insulin cascade. These data add a new dimension to existing models and position Drosophila as a powerful tool to study the relation between dietary lipid cues and the insulin-induced cellular signal pathway.

Monday, December 30th - Disease Models

Si, H., Ma, P., Liang, Q., Yin, Y., Wang, P., Zhang, Q., Wang, S. and Deng, H. (2019). Overexpression of pink1 or parkin in indirect flight muscles promotes mitochondrial proteostasis and extends lifespan in Drosophila melanogaster. PLoS One 14(11): e0225214. PubMed ID: 31714929
Summary:
Dysfunctional mitochondria have been implicated in aging and age-related disorders such as Parkinson's diseases (PD). Previous work has shown that pink1 and parkin, two familial PD genes, function in a linear pathway to maintain mitochondrial integrity and function. Studies of mammalian cell lines also suggest that these genes regulate mitochondrial autophagy(mitophagy). Overexpressing Parkin promotes proteostasis and function of aged muscles both in fruit flies and mice, and recent studies also indicated that mitochondrial ubiquitination are accumulated in aged muscles. However, the underlying mechanisms for pink1 and parkin mediated mitophagy on longevity is not fully understood. This study found that mitochondrial ubiquitination increased in indirect flight muscles (IFMs) in an age-dependent manner. Overexpression of pink1 or parkin in IFMs can abolish mitochondrial ubiquitination, restore ATP level and extend lifespan, while blocking autophagy via ATG1 knock-down suppress these effects in aged IFMs. Taken together, these results show that pink1/parkin promotes mitophagy of mitochondrial ubiquitination in aged muscles and extend lifespan in an Atg1-dependent manner. This study provides physiological evidence that mitophagy of mitochondrial ubiquitination mediated by PINK1/ Parkin is crucial for muscle function and highlights the role of mitophagy in the pathogenesis of chronic diseases like PD.
Shimada, S., Muraoka, Y., Ibaraki, K., Takano-Shimizu-Kouno, T., Yoshida, H. and Yamaguchi, M. (2019). Identification of CR43467 encoding a long non-coding RNA as a novel genetic interactant with dFIG4, a CMT-causing gene. Exp Cell Res: 111711. PubMed ID: 31704059
Summary:
The eye imaginal disc-specific knockdown of dFIG4, a Drosophila homolog of FIG4 that is one of the Charcot-Marie-Tooth disease (CMT)-causing genes, induces an aberrant adult compound eye morphology, the so-called rough eye phenotype. A previous modifier screening on the dFIG4 knockdown-induced rough eye phenotype identified several genes, including CR18854, encoding a long non-coding RNA (lncRNA), as genetic interactants with dFIG4. The present study, in more extensive genetic screening, found that the deletion of a gene locus encoding both Odorant rector 46a (Or46a) and lncRNA CR43467 effectively suppressed the rough eye phenotype induced by the knockdown of dFIG4. Both genes were located on the same locus, but oriented in opposite directions. In order to identify which of these genes is responsible for the suppression of the rough eye phenotype, a CR43467-specific knockdown line was established using the CRISPR-dCas9 system. By using this system, it was demonstrated that the CR43467 gene, but not the Or46a gene, genetically interacted with the dFIG4 gene. The knockdown of CR43467 rescued the reductions in the length of synaptic branches and number of boutons at neuromuscular junctions induced by the knockdown of dFIG4. The vacuole enlargement phenotype induced by the fat body-specific dFIG4 knockdown was also effectively suppressed by the knockdown of CR43467. This study has identified another gene encoding lncRNA, CR43467 as a genetic interactant with the CMT-causing gene.
Lin, Y. H., Maaroufi, H. O., Ibrahim, E., Kucerova, L. and Zurovec, M. (2019). Expression of human mutant Huntingtin protein in Drosophila hemocytes impairs immune responses. Front Immunol 10: 2405. PubMed ID: 31681295
Summary:
The pathogenic effect of mutant HTT (mHTT) which causes Huntington disease (HD) are not restricted to nervous system. Such phenotypes include aberrant immune responses observed in the HD models. However, it is still unclear how this immune dysregulation influences the innate immune response against pathogenic infection. This study used transgenic Drosophila melanogaster expressing mutant HTT protein (mHTT) with hemocyte-specific drivers and examined the immune responses and hemocyte function. mHTT expression in the hemocytes did not affect fly viability, but the numbers of circulating hemocytes were significantly decreased. Consequently, it was observed that the expression of mHTT in the hemocytes compromised the immune responses including clot formation and encapsulation which lead to the increased susceptibility to entomopathogenic nematode and parasitoid wasp infections. In addition, mHTT expression in Drosophila macrophage-like S2 cells in vitro reduced ATP levels, phagocytic activity and the induction of antimicrobial peptides. Further effects observed in mHTT-expressing cells included the altered production of cytokines and activation of JAK/STAT signaling. The present study shows that the expression of mHTT in Drosophila hemocytes causes deficient cellular and humoral immune responses against invading pathogens. These findings provide the insight into the pathogenic effects of mHTT in the immune cells.
Millet-Boureima, C., Chingle, R., Lubell, W. D. and Gamberi, C. (2019). Cyst Reduction in a Polycystic Kidney Disease Drosophila Model Using Smac Mimics. Biomedicines 7(4). PubMed ID: 31635379
Summary:
Autosomal dominant polycystic kidney disease (ADPKD) is an inherited malady affecting 12.5 million people worldwide. Therapeutic options to treat PKD are limited, due in part to lack of precise knowledge of underlying pathological mechanisms. Mimics of the second mitochondria-derived activator of caspases (Smac), a mitochondrial protein that is released together with cytochrome c from the mitochondria during apoptosis, have exhibited activity as antineoplastic agents and reported recently to ameliorate cysts in a murine ADPKD model, possibly by differentially targeting cystic cells and sparing the surrounding tissue. A first-in-kind Drosophila PKD model has now been employed to probe further the activity of novel Smac mimics. Substantial reduction of cystic defects was observed in the Malpighian (renal) tubules of treated flies, underscoring mechanistic conservation of the cystic pathways and potential for efficient testing of drug prototypes in this PKD model. Moreover, the observed differential rescue of the anterior and posterior tubules overall, and within their physiologically diverse intermediate and terminal regions implied a nuanced response in distinct tubular regions contingent upon the structure of the Smac mimic. Knowledge gained from studying Smac mimics reveals the capacity for the Drosophila model to precisely probe PKD pharmacology highlighting the value for such critical evaluation of factors implicated in renal function and pathology.
Pragati, Chanu, S. I. and Sarkar, S. (2019). Reduced expression of dMyc mitigates Tau(V337M) mediated neurotoxicity by preventing the Tau hyperphosphorylation and inducing autophagy in Drosophila. Neurosci Lett: 134622. PubMed ID: 31715291
Summary:
Tauopathies such as Alzheimer's disease (AD), Pick's disease (PiD), Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) etc. represent a group of age-related neurodegenerative disorders in which tau protein loses its normal conformation mostly due to hyperphosphorylation and subsequent formation of the aggregates of defined shapes, known as Neurofibrillary Tangles (NFTs). Earlier work has demonstrated that reduced dosage of dmyc (Drosophila homolog of human cmyc proto-oncogene) restricts tau(WT) mediated disease pathogenesis by regulating the phosphorylation status of tau. This study demonstrates further that the downregulation of dmyc also alleviates the mutant human-tau [tau(V337M)] mediated neurotoxicity in Drosophila by improving disease defects. Moreover, tissue-specific downregulation of dmyc also induces cellular autophagy which facilitates the disposal of misfolded proteins via lysosome-mediated proteostasis. These findings demonstrate the capability of dmyc in the suppression of different forms of human tauopathies in Drosophila disease models. Interestingly, due to the conserved characteristics of dmyc/cmyc across the animal kingdom, this study strengthens the possibility of utilizing this gene as an effective drug target against tauopathies.
Schroeder, A. M., Allahyari, M., Vogler, G., Missinato, M. A., Nielsen, T., Yu, M. S., Theis, J. L., Larsen, L. A., Goyal, P., Rosenfeld, J., Nelson, T. J., Olson, T. M., Colas, A. R., Grossfeld, P. and Bodmer, R. (2019). Model system identification of novel congenital heart disease gene candidates: focus on RPL13. Hum Mol Genet. PubMed ID: 31625562
Summary:
Understanding of the genetic players and networks involved in congenital heart disease (CHD) pathogenesis is limited. This study searched for de novo Copy Number Variations (CNVs) in a cohort of 167 CHD patients to identify DNA segments containing potential pathogenic genes. An integrated high-throughput phenotypical platform was developed to probe for defects in cardiogenesis and cardiac output in human iPSC-derived multipotent cardiac progenitor cells (MCPs) and, in parallel, in the Drosophila in vivo heart model. Notably, knockdown in MCPs of RPL13, a ribosomal gene and SON, an RNA splicing cofactor, reduced proliferation and differentiation of cardiomyocytes, while increasing fibroblasts. In the fly, heart-specific RpL13 knockdown, predominantly at embryonic stages, resulted in a striking 'no heart' phenotype. Knockdown of Son and Pdss2, among others, caused structural and functional defects, including reduced or abolished contractility, respectively. This study has identified new genes as candidates for causing human congenital heart disease, with particular emphasis on ribosomal genes, such as RPL13. This approach will allow for testing large numbers of CHD candidates, based on patient genomic data, and for building upon existing genetic networks involved in heart development and disease.

Friday, December 27th - RNA

Harrison, J. U., Parton, R. M., Davis, I. and Baker, R. E. (2019). Testing Models of mRNA Localization Reveals Robustness Regulated by Reducing Transport between Cells. Biophys J. PubMed ID: 31708163
Summary:
Robust control of gene expression in both space and time is of central importance in the regulation of cellular processes and for multicellular development. However, the mechanisms by which robustness is achieved are generally not identified or well understood. For example, messenger RNA (mRNA) localization by molecular motor-driven transport is crucial for cell polarization in numerous contexts, but the regulatory mechanisms that enable this process to take place in the face of noise or significant perturbations are not fully understood. This study used a combined experimental-theoretical approach to characterize the robustness of Gurken/transforming growth factor-alpha mRNA localization in Drosophila egg chambers, where the oocyte and 15 surrounding nurse cells are connected in a stereotypic network via intracellular bridges known as ring canals. A mathematical model was constructed that encodes simplified descriptions of the range of steps involved in mRNA localization, including production and transport between and within cells until the final destination in the oocyte. Using Bayesian inference, this model was calibrated using quantitative single molecule fluorescence in situ hybridization data. By analyzing both the steady state and dynamic behaviors of the model, estimates are provided for the rates of different steps of the localization process as well as the extent of directional bias in transport through the ring canals. The model predicts that mRNA synthesis and transport must be tightly balanced to maintain robustness, a prediction that was tested experimentally using an overexpression mutant. Surprisingly, the overexpression mutant fails to display the anticipated degree of overaccumulation of mRNA in the oocyte predicted by the model. Through careful model-based analysis of quantitative data from the overexpression mutant, evidence is shown of saturation of the transport of mRNA through ring canals. It is concluded that this saturation engenders robustness of the localization process in the face of significant variation in the levels of mRNA synthesis.
Dzaki, N., Woo, W. K., Thangadurai, S. and Azzam, G. (2019). MicroRNA regulation of CTP synthase and cytoophidium in Drosophila melanogaster. Exp Cell Res 385(2): 111688. PubMed ID: 31678212
Summary:
CTPsyn is a crucial metabolic enzyme which synthesizes CTP nucleotides. It has the extraordinary ability to compartmentalize into filaments termed cytoophidia. Though the structure is evolutionarily conserved across kingdoms, the mechanisms behind their formation remain unknown. MicroRNAs (miRNAs) are short single-stranded RNA capable of directing mRNA silencing and degradation. D. melanogaster has a high total gene count to miRNA gene number ratio, alluding to the possibility that CTPsyn too may come under their regulation. A thorough miRNA overexpression involving 123 miRNAs was conducted, followed by CTPsyn-specific staining upon cytoophidia-rich egg chambers. This revealed a small group of candidates which confer either a lengthening or truncating effect on cytoophidia, suggesting they may play a role in regulating CTPsyn. MiR-975 and miR-1014 are both cytoophidia-elongating, whereas miR-190 and miR-932 are cytoophidia-shortening. Though target prediction shows that miR-975 and miR-932 do indeed have binding sites on CTPsyn mRNA, in vitro assays instead revealed a low probability of this being true, instead indicating that the effects asserted by overexpressed miRNAs indirectly reach CTPsyn and its cytoophidia through the actions of middling elements. In silico target prediction and qPCR quantification indicated that, at least for miR-932 and miR-1014, these undetermined elements may be players in fat metabolism. This is the first study to thoroughly investigate miRNAs in connection to CTPsyn expression and activity in any species. The findings presented could serve as a basis for further queries into not only the fundamental aspects of the enzyme's regulation, but may uncover new facets of closely related pathways as well.
Foley, L., Ling, J., Joshi, R., Evantal, N., Kadener, S. and Emery, P. (2019). Drosophila PSI controls circadian period and the phase of circadian behavior under temperature cycle via tim splicing. Elife 8. PubMed ID: 31702555
Summary:
The Drosophila circadian pacemaker consists of transcriptional feedback loops subjected to post-transcriptional and post-translational regulation. While post-translational regulatory mechanisms have been studied in detail, much less is known about circadian post-transcriptional control. Thus, this study targeted 364 RNA binding and RNA associated proteins with RNA interference. Among the 43 hits that were identified was the alternative splicing regulator P-element somatic inhibitor (PSI). PSI regulates the thermosensitive alternative splicing of timeless (tim), promoting splicing events favored at warm temperature over those increased at cold temperature. Psi downregulation shortens the period of circadian rhythms and advances the phase of circadian behavior under temperature cycle. Interestingly, both phenotypes were suppressed in flies that could produce TIM proteins only from a transgene that cannot form the thermosensitive splicing isoforms. Therefore, it is concluded that PSI regulates the period of Drosophila circadian rhythms and circadian behavior phase during temperature cycling through its modulation of the tim splicing pattern.
Gu, P., Gong, J., Shang, Y., Wang, F., Ruppell, K. T., Ma, Z., Sheehan, A. E., Freeman, M. R. and Xiang, Y. (2019). Polymodal nociception in Drosophila requires alternative splicing of TrpA1. Curr Biol. PubMed ID: 31735672
Summary:
Transcripts of noxious stimulus-detecting TrpA1 channels are alternatively spliced. Despite the importance of nociception for survival, the in vivo significance of expressing different TrpA1 isoforms is largely unknown. This study developed a novel genetic approach to generate Drosophila knockin strains expressing single TrpA1 isoforms. Drosophila TrpA1 mediates heat and UVC-triggered nociception. TrpA1-C and TrpA1-D, two alternative isoforms, were found to be co-expressed in nociceptors. When examined in heterologous cells, both TrpA1-C and TrpA1-D are activated by heat and UVC. By contrast, analysis of knockin flies reveals the striking functional specificity; TrpA1-C mediates UVC-nociception, whereas TrpA1-D mediates heat-nociception. Therefore, in vivo functions of TrpA1-C and TrpA1-D are different from each other and are different from their in vitro properties. These results indicate that a given sensory stimulus preferentially activates a single TrpA1 isoform in vivo and that polymodal nociception requires co-expression of TrpA1 isoforms, providing novel insights of how alternative splicing regulates nociception.
Martin Anduaga, A., Evantal, N., Patop, I. L., Bartok, O., Weiss, R. and Kadener, S. (2019). Thermosensitive alternative splicing senses and mediates temperature adaptation in Drosophila. Elife 8. PubMed ID: 31702556
Summary:
Circadian rhythms are generated by cyclic transcription, translation, and degradation of clock gene products, including timeless (tim), but how the circadian clock senses and adapts to temperature changes is not completely understood. This study showed that temperature dramatically changes the splicing pattern of tim in Drosophila. At 18 ° C, TIM levels are low due to the induction of two cold-specific isoforms: tim-cold and tim-short&cold. At 29 ° C, another isoform, tim-medium, is upregulated. This isoform switching regulates the levels and activity of TIM as each isoform has a specific function. tim-short&cold was found to encode a protein that rescues the behavioral defects of tim01 mutants and that flies in which tim-short&cold is abrogated have abnormal locomotor activity. In addition, miRNA-mediated control limits the expression of some of these isoforms. Finally, the data using minigenes suggest that tim alternative splicing might act as a thermometer for the circadian clock.
Sechi, S., Frappaolo, A., Karimpour-Ghahnavieh, A., Gottardo, M., Burla, R., Di Francesco, L., Szafer-Glusman, E., Schinina, E., Fuller, M. T., Saggio, I., Riparbelli, M. G., Callaini, G. and Giansanti, M. G. (2019). Drosophila Doublefault protein coordinates multiple events during male meiosis by controlling mRNA translation. Development 146(22). PubMed ID: 31645358
Summary:
During the extended prophase of Drosophila gametogenesis, spermatocytes undergo robust gene transcription and store many transcripts in the cytoplasm in a repressed state, until translational activation of select mRNAs in later steps of spermatogenesis. This study characterized the Drosophila Doublefault (Dbf) protein as a C2H2 zinc-finger protein, primarily expressed in testes, that is required for normal meiotic division and spermiogenesis. Loss of Dbf causes premature centriole disengagement and affects spindle structure, chromosome segregation and cytokinesis. Dbf interacts with the RNA-binding protein Syncrip/hnRNPQ, a key regulator of localized translation in Drosophila. It is proposed that the pleiotropic effects of dbf loss-of-function mutants are associated with the requirement of dbf function for translation of specific transcripts in spermatocytes. In agreement with this hypothesis, Dbf protein binds cyclin B mRNA and is essential for translation of cyclin B in mature spermatocytes.

Thursday, December 26th - Evolution

Schroeder, C. M., Valenzuela, J. R., Natividad, I. M., Hocky, G. M. and Malik, H. S. (2019). A burst of genetic innovation in Drosophila actin-related proteins for testis-specific function. Mol Biol Evol. PubMed ID: 31697328
Summary:
Many cytoskeletal proteins perform fundamental biological processes and are evolutionarily ancient. For example, the superfamily of actin-related proteins (Arps) specialized early in eukaryotic evolution for diverse cellular roles in the cytoplasm and the nucleus. Despite its strict conservation across eukaryotes, this study found that the Arp superfamily has undergone dramatic lineage-specific diversification in Drosophila. Phylogenomic analyses reveal four independent Arp gene duplications that occurred in the common ancestor of the obscura group of Drosophila and have been mostly preserved in this lineage. All four obscura-specific Arp paralogs are predominantly expressed in the male germline and have evolved under positive selection. The analyses focussed on the divergent Arp2D paralog, which arose via a retroduplication event from Arp2, a component of the Arp2/3 complex that polymerizes branched actin networks. Computational modeling analyses suggests that Arp2D can replace Arp2 in the Arp2/3 complex and bind actin monomers. Together with the signature of positive selection, these findings suggest that Arp2D may augment Arp2's functions in the male germline. Indeed, it was found that Arp2D is expressed during and following male meiosis, where it localizes to distinct locations such as actin cones-specialized cytoskeletal structures that separate bundled spermatids into individual mature sperm. It is hypothesized that this unprecedented burst of genetic innovation in cytoskeletal proteins may have been driven by the evolution of sperm heteromorphism in the obscura group of Drosophila.
Wei, K. H. and Bachtrog, D. (2019). Ancestral male recombination in Drosophila albomicans produced geographically restricted neo-Y chromosome haplotypes varying in age and onset of decay. PLoS Genet 15(11): e1008502. PubMed ID: 31738748
Summary:
Male Drosophila typically have achiasmatic meiosis, and fusions between autosomes and the Y chromosome have repeatedly created non-recombining neo-Y chromosomes that degenerate. Intriguingly, Drosophila nasuta males recombine, but their close relative D. albomicans reverted back to achiasmy after evolving neo-sex chromosomes. This study used genome-wide polymorphism data to reconstruct the complex evolutionary history of neo-sex chromosomes in D. albomicans and examine the effect of recombination and its cessation on the initiation of neo-Y decay. Population and phylogenomic analyses reveal three distinct neo-Y types that are geographically restricted. Due to ancestral recombination with the neo-X, overall nucleotide diversity on the neo-Y is similar to the neo-X but severely reduced within neo-Y types. Consistently, the neo-Y chromosomes fail to form a monophyletic clade in sliding window trees outside of the region proximal to the fusion. Based on tree topology changes, the recombination breakpoints were infered that produced haplotypes specific to each neo-Y type. Recombination became suppressed at different time points for the different neo-Y haplotypes. Haplotype age correlates with onset of neo-Y decay, and older neo-Y haplotypes show more fixed gene disruption via frameshift indels and down-regulation of neo-Y alleles. Genes are downregulated independently on the different neo-Ys, but are depleted of testes-expressed genes across all haplotypes. This indicates that genes important for male function are initially shielded from degeneration. Thesee results offer a time course of the early progression of Y chromosome evolution, showing how the suppression of recombination, through the reversal to achiasmy in D. albomicans males, initiates the process of degeneration.
Chakraborty, M., Emerson, J. J., Macdonald, S. J. and Long, A. D. (2019). Structural variants exhibit widespread allelic heterogeneity and shape variation in complex traits. Nat Commun 10(1): 4872. PubMed ID: 31653862
Summary:
It has been hypothesized that individually-rare hidden structural variants (SVs) could account for a significant fraction of variation in complex traits. This study identified more than 20,000 euchromatic SVs from 14 Drosophila melanogaster genome assemblies, of which ~40% are invisible to high specificity short-read genotyping approaches. SVs are common, with 31.5% of diploid individuals harboring a SV in genes larger than 5kb, and 24% harboring multiple SVs in genes larger than 10kb. SV minor allele frequencies are rarer than amino acid polymorphisms, suggesting that SVs are more deleterious. A number of functionally important genes were shown to harbor previously hidden structural variants likely to affect complex phenotypes. Furthermore, SVs are overrepresented in candidate genes associated with quantitative trait loci mapped using the Drosophila Synthetic Population Resource. It is concluded that SVs are ubiquitous, frequently constitute a heterogeneous allelic series, and can act as rare alleles of large effect.
Wong, H. W. S. and Holman, L. (2019). Fitness consequences of the selfish supergene Segregation Distorter. J Evol Biol. PubMed ID: 31605400
Summary:
Segregation distorters are selfish genetic elements that subvert Mendelian inheritance, for example by destroying gametes that do not carry the distorter. Simple theoretical models predict that distorter alleles will either spread to fixation, or stabilise at some high intermediate frequency. However, many distorters have substantially lower allele frequencies than predicted by simple models, suggesting that key sources of selection remain to be discovered. This study measured the fitness of Drosophila melanogaster adults and juveniles carrying zero, one, or two copies of three different variants of the naturally-occurring supergene Segregation Distorter (SD), in order to investigate why SD alleles remain relatively rare within populations despite being preferentially inherited. First, it was shown that the three SD variants differ in the severity and dominance of the fitness costs they impose on individuals carrying them. Second, SD-carrying parents produced less fit offspring in some crosses, independent of of spring genotype, indicating that SD alleles can have non-genetic, transgenerational costs in addition to their direct costs. Third, it was found that SD carriers sometimes produce a biased offspring sex ratio, perhaps due to off-target effects of SD on the sex chromosomes.
Keaney, T. A., Wong, H. W. S., Dowling, D. K., Jones, T. M. and Holman, L. (2019). Mother's curse and indirect genetic effects: Do males matter to mitochondrial genome evolution?. J Evol Biol. PubMed ID: 31650630
Summary:
Maternal inheritance of mitochondrial DNA (mtDNA) was originally thought to prevent any response to selection on male phenotypic variation attributable to mtDNA, resulting in a male-biased mtDNA mutation load ("mother's curse"). However, the theory underpinning this claim implicitly assumes that a male's mtDNA has no effect on the fitness of females he comes into contact with. If such "mitochondrially encoded indirect genetics effects" (mtIGEs) do in fact exist, and there is relatedness between the mitochondrial genomes of interacting males and females, male mtDNA-encoded traits can undergo adaptation after all. This possibility was tested using strains of Drosophila melanogaster that differ in their mtDNA. The experiments indicate that female fitness is influenced by the mtDNA carried by males that the females encounter, which could plausibly allow the mitochondrial genome to evolve via kin selection. It is argued that mtIGEs are probably common, and that this might ameliorate or exacerbate mother's curse.
Little, C. M., Rizzato, A. R., Charbonneau, L., Chapman, T. and Hillier, N. K. (2019). Color preference of the spotted wing Drosophila, Drosophila suzukii. Sci Rep 9(1): 16051. PubMed ID: 31690772
Summary:
Drosophila suzukii Matsumura (Diptera: Drosophilidae) is a significant invasive pest in soft-skin fruits and berries in Asia, Europe, and North and South America. Many herbivorous insects use multiple cues for host selection, particularly olfactory and visual stimuli. The visual system of closely-related Drosophila melanogaster is well-documented, expressing strong sensitivity to short-wavelength colors (ultraviolet to green) and only limited sensitivity to long-wavelength colors (red to infrared). The results suggest that D. suzukii have limited ability to distinguish red consistent with visual sensitivity range within the melanogaster subgroup. It is proposed that color contrast rather than color appearance may be of greater importance in orientation and attraction. It is proposed that differences in reflectance between light wavelengths important for color opponency are key to color discrimination to provide color contrast between foreground and background, as occurs between fruit and foliage, during host-finding.

Monday, December 23rd - Larval and Adult Physiology

Liu, Y., Bao, H., Wang, W. and Lim, H. Y. (2019). Cardiac Snail family of transcription factors directs systemic lipid metabolism in Drosophila. PLoS Genet 15(11): e1008487. PubMed ID: 31725726
Summary:
Maintenance of normal lipid homeostasis is crucial to heart function. On the other hand, the heart is now recognized to serve an important role in regulating systemic lipid metabolism; however, the molecular basis remains unclear. This study identified the Drosophila Snail family of transcription factors (herein termed Sna TFs) as new mediators of the heart control of systemic lipid metabolism. Overexpression of Sna TF genes specifically in the heart promotes whole-body leanness whereas their knockdown in the heart promotes obesity. In addition, flies that are heterozygous for a snail deficiency chromosome also exhibit systemic obesity, and cardiac-specific overexpression of Sna substantially reverses systemic obesity in these flies. It was further shown that genetically manipulating Sna TF levels in the fat body and intestine do not affect systemic lipid levels. Mechanistically, this study found that flies bearing the overexpression or inhibition of Sna TFs in the postnatal heart exhibit only systemic lipid metabolic defects but not heart abnormalities. Cardiac-specific alterations of Sna TF levels also do not perturb cardiac morphology, viability, lipid metabolism or fly food intake. On the other hand, cardiac-specific manipulations of Sna TF levels alter lipogenesis and lipolysis gene expression, mitochondrial biogenesis and respiration, and lipid storage droplet 1 and 2 (Lsd-1 and Lsd-2) levels in the fat body. Together, these results reveal a novel and specific role of Sna TFs in the heart on systemic lipid homeostasis maintenance that is independent of cardiac development and function and involves the governance of triglyceride synthesis and breakdown, energy utilization, and lipid droplet dynamics in the fat body.
Lee, D. C., et al. (2019). Dietary supplementation with the ketogenic diet metabolite beta-hydroxybutyrate ameliorates post-TBI aggression in young-adult male Drosophila. Front Neurosci 13: 1140. PubMed ID: 31736687
Summary:
Traumatic brain injury (TBI), caused by repeated concussive head trauma can induce chronic traumatic encephalopathy (CTE), a neurodegenerative disease featuring behavioral symptoms ranging from cognitive deficits to elevated aggression. In a Drosophila model, this study used a high-impact trauma device to induce TBI-like symptoms and to study post-TBI behavioral outcomes. Following TBI, aggression in banged male flies was significantly elevated as compared with that in unbanged flies. Various forms of dietary therapy, especially the high-fat, low-carbohydrate ketogenic diet (KD), have recently been explored for a wide variety of neuropathies. It is thus hypothesized that putatively neuroprotective dietary interventions might be able to suppress post-traumatic elevations in aggressive behavior in animals subjected to head-trauma-inducing strikes, or "bangs". A normal high-carbohydrate Drosophila diet was supplemented with the KD metabolite beta-hydroxybutyrate (beta-HB)-a ketone body (KB). Banged flies raised on a KB-supplemented diet exhibited a marked reduction in aggression, whereas aggression in unbanged flies was equivalent whether dieted with KB supplements or not. Pharmacological blockade of the ATP-sensitive potassium (KATP) channel abrogated KB effects reducing post-TBI aggression while pharmacological activation mimicked them, suggesting a mechanism by which KBs act in this model. KBs did not significantly extend lifespan in banged flies, but markedly extended lifespan in unbanged flies. This study has developed a functional model for the study of post-TBI elevations of aggression. Further, It is concluded that dietary interventions may be a fruitful avenue for further exploration of treatments for TBI- and CTE-related cognitive-behavioral symptoms.
Schonborn, J. W., Jehrke, L., Mettler-Altmann, T. and Beller, M. (2019). FlySilico: Flux balance modeling of Drosophila larval growth and resource allocation. Sci Rep 9(1): 17156. PubMed ID: 31748517
Summary:
Organisms depend on a highly connected and regulated network of biochemical reactions fueling life sustaining and growth promoting functions. While details of this metabolic network are well established, knowledge of the superordinate regulatory design principles is limited. This study investigated by iterative wet lab and modeling experiments the resource allocation process during the larval development of Drosophila melanogaster. This system was chosen, as survival of the animals depends on the successful allocation of their available resources to the conflicting processes of growth and storage metabolite deposition. First, "FlySilico", a curated metabolic network of Drosophila was generated, and time-resolved growth and metabolite measurements were performed with larvae raised on a holidic diet. Subsequently, flux balance analysis simulations were performed, and the predictive power of this model was tested by simulating the impact of diet alterations on growth and metabolism. The predictions of this study correctly identified the essential amino acids as growth limiting factor, and metabolic flux differences in agreement with experimental data. Thus, this study presents a framework to study important questions of resource allocation in a multicellular organism including process priorization and optimality principles.
Livingston, D. B. H., Patel, H., Donini, A. and MacMillan, H. A. (2020). Active transport of brilliant blue FCF across the Drosophila midgut and Malpighian tubule epithelia. Comp Biochem Physiol A Mol Integr Physiol 239: 110588. PubMed ID: 31648063
Summary:
Under conditions of stress, many animals suffer from epithelial barrier disruption that can cause molecules to leak down their concentration gradients, potentially causing a loss of organismal homeostasis, further injury or death. Drosophila is a common insect model, used to study barrier disruption related to aging, traumatic injury, or environmental stress. Net leak of a non-toxic dye (Brilliant blue FCF) from the gut lumen to the hemolymph is often used to identify barrier failure under these conditions, but Drosophila are capable of actively transporting structurally-similar compounds. This study examined whether cold stress (like other stresses) causes Brilliant blue FCF (BB-FCF) to appear in the hemolymph of flies fed the dye, and if so whether Drosophila are capable of clearing this dye from their body following chilling. Using in situ midgut leak and transport assays as well as Ramsay assays of Malpighian tubule transport, this study tested whether these ionoregulatory epithelia can actively transport BB-FCF. In doing so, it was found that the Drosophila midgut and Malpighian tubules can mobilize BB-FCF via an active transcellular pathway, suggesting that elevated concentrations of the dye in the hemolymph may occur from increased paracellular permeability, reduced transcellular clearance, or both. It is concluded that Drosophila are able to actively secrete Brilliant blue FCF, a commonly used marker of barrier dysfunction.
Lee, S., Jones, W. D. and Kim, D. H. (2019). A cyclic nucleotide-gated channel in the brain regulates olfactory modulation through neuropeptide F in fruit fly Drosophila melanogaster. Arch Insect Biochem Physiol: e21620. PubMed ID: 31625196
Summary:
Olfactory sensing and its modulation are important for the insects in recognizing diverse odors from the environment and in making correct decisions to survive. Identifying new genes involved in olfactory modulation and unveiling their mechanisms may lead to understanding of decision making processes in the central nervous system. This study reports a novel olfactory function of the cyclic nucleotide-gated (CNG) channel CG42260 in modulating ab3A antennal olfactory sensory neurons, which specifically respond to food-derived odors in fruit fly Drosophila melanogaster. Two independent CG42260 mutants were found to show reduced responses in the ab3A neurons. Unlike mammalian CNGs, CG42260 is not expressed in the odorant sensory neurons but broadly in the central nervous system including neuropeptide-producing cells. By using molecular genetic tools, this study identified CG42260 expression in one pair of neuropeptide F (NPF) positive L1-l cells known to modulate food odor responsiveness. Knockdown of CG42260 in the NPF neurons reduced production of NPF in Ll-1 cells, which in turn, led to reduction of neuronal responses of the ab3A neurons. These findings show the novel biological function of CG42260 in modulating olfactory responses to food odor through NPF.
Nash, T. R., Chow, E. S., Law, A. D., Fu, S. D., Fuszara, E., Bilska, A., Bebas, P., Kretzschmar, D. and Giebultowicz, J. M. (2019). Daily blue-light exposure shortens lifespan and causes brain neurodegeneration in Drosophila. NPJ Aging Mech Dis 5: 8. PubMed ID: 31636947
Summary:
Light is necessary for life, but prolonged exposure to artificial light is a matter of increasing health concern. Humans are exposed to increased amounts of light in the blue spectrum produced by light-emitting diodes (LEDs), which can interfere with normal sleep cycles. The LED technologies are relatively new; therefore, the long-term effects of exposure to blue light across the lifespan are not understood. This study investigated the effects of light in the model organism, Drosophila melanogaster, and determined that flies maintained in daily cycles of 12-h blue LED and 12-h darkness had significantly reduced longevity compared with flies maintained in constant darkness or in white light with blue wavelengths blocked. Exposure of adult flies to 12 h of blue light per day accelerated aging phenotypes causing damage to retinal cells, brain neurodegeneration, and impaired locomotion. Brain damage and locomotor impairments do not depend on the degeneration in the retina, as these phenotypes were evident under blue light in flies with genetically ablated eyes. Blue light induces expression of stress-responsive genes in old flies but not in young, suggesting that cumulative light exposure acts as a stressor during aging. This study also determined that several known blue-light-sensitive proteins are not acting in pathways mediating detrimental light effects. This study reveals the unexpected effects of blue light on fly brain and establishes Drosophila as a model in which to investigate long-term effects of blue light at the cellular and organismal level.

Friday, December 20th - Behavior

Bertolini, E., Schubert, F. K., Zanini, D., Sehadova, H., Helfrich-Forster, C. and Menegazzi, P. (2019). Life at high latitudes does not require circadian behavioral rhythmicity under constant darkness. Curr Biol 29(22): 3928-3936. PubMed ID: 31679928
Summary:
Nearly all organisms evolved endogenous self-sustained timekeeping mechanisms to track and anticipate cyclic changes in the environment. Circadian clocks, with a periodicity of about 24 h, allow animals to adapt to day-night cycles. Biological clocks are highly adaptive, but strong behavioral rhythms might be a disadvantage for adaptation to weakly rhythmic environments such as polar areas. Several high-latitude species, including Drosophila species, were found to be highly arrhythmic under constant conditions. Furthermore, Drosophila species from subarctic regions can extend evening activity until dusk under long days. These traits depend on the clock network neurochemistry, and it has been previously proposed that high-latitude Drosophila species evolved specific clock adaptations to colonize polar regions. This study broadened this analysis to 3 species of the Chymomyza genus, which diverged circa 5 million years before the Drosophila radiation and colonized both low and high latitudes. C. costata, pararufithorax, and procnemis, independently of their latitude of origin, possess the clock neuronal network of low-latitude Drosophila species, and their locomotor activity does not track dusk under long photoperiods. Nevertheless, the high-latitude C. costata becomes arrhythmic under constant darkness (DD), whereas the two low-latitude species remain rhythmic. Different mechanisms are behind the arrhythmicity in DD of C. costata and the high-latitude Drosophila ezoana, suggesting that the ability to maintain behavioral rhythms has been lost more than once during drosophilid evolution and that it might indeed be an evolutionary adaptation for life at high latitudes.
Fowler, E. K., Bradley, T., Moxon, S. and Chapman, T. (2019). Divergence in transcriptional and regulatory responses to mating in male and female fruitflies. Sci Rep 9(1): 16100. PubMed ID: 31695054
Summary:
Mating induces extensive physiological, biochemical and behavioural changes in female animals of many taxa. In contrast, the overall phenotypic and transcriptomic consequences of mating for males, hence how they might differ from those of females, are poorly described. Post mating responses in each sex are rapidly initiated, predicting the existence of regulatory mechanisms in addition to transcriptional responses involving de novo gene expression. That post mating responses appear different for each sex also predicts that the genome-wide signatures of mating should show evidence of sex-specific specialisation. This study used high resolution RNA sequencing to provide the first direct comparisons of the transcriptomic responses of male and female Drosophila to mating, and the first comparison of mating-responsive miRNAs in both sexes in any species. As predicted, the results revealed the existence of sex- and body part-specific mRNA and miRNA expression profiles. More genes were differentially expressed in the female head-thorax than the abdomen following mating, whereas the opposite was true in males. Indeed, the transcriptional profile of male head-thorax tissue was largely unaffected by mating, and no differentially expressed genes were detected at the most stringent significance threshold. A subset of ribosomal genes in females were differentially expressed in both body parts, but in opposite directions, consistent with the existence of body part-specific resource allocation switching. Novel, mating-responsive miRNAs in each sex were also identified, and a miRNA-mRNA interactions analysis revealed putative targets among mating-responsive genes. This study has shown th structure of genome-wide responses by each sex to mating is strongly divergent, and provide new insights into how shared genomes can achieve characteristic distinctiveness.
Cazale-Debat, L., Houot, B., Farine, J. P., Everaerts, C. and Ferveur, J. F. (2019). Flying Drosophila show sex-specific attraction to fly-labelled food. Sci Rep 9(1): 14947. PubMed ID: 31628403
Summary:
Animals searching for food and sexual partners often use odourant mixtures combining food-derived molecules and pheromones. For orientation, the vinegar fly Drosophila melanogaster uses three types of chemical cues: (i) the male volatile pheromone 11-cis-vaccenyl acetate (cVA), (ii) sex-specific cuticular hydrocarbons (CHs; and CH-derived compounds), and (iii) food-derived molecules resulting from microbiota activity. To evaluate the effects of these chemicals on odour-tracking behaviour, Drosophila individuals were tested in a wind tunnel. Upwind flight and food preference were measured in individual control males and females presented with a choice of two food sources labelled by fly lines producing varying amounts of CHs and/or cVA. The flies originated from different species or strains, or their microbiota was manipulated. The following was found: (i) fly-labelled food could attract-but never repel-flies; (ii) the landing frequency on fly-labelled food was positively correlated with an increased flight duration; (iii) male-but not female or non-sex-specific-CHs tended to increase the landing frequency on fly-labelled food; (iv) cVA increased female-but not male-preference for cVA-rich food; and (v) microbiota-derived compounds only affected male upwind flight latency. Therefore, sex pheromones interact with food volatile chemicals to induce sex-specific flight responses in Drosophila.
Ferdenache, M., Bezzar-Bendjazia, R., Marion-Poll, F. and Kilani-Morakchi, S. (2019). Transgenerational effects from single larval exposure to azadirachtin on life history and behavior traits of Drosophila melanogaster. Sci Rep 9(1): 17015. PubMed ID: 31745147
Summary:
Azadirachtin is one of the successful botanical pesticides in agricultural use with a broad-spectrum insecticide activity, but its possible transgenerational effects have not been under much scrutiny. The effects of sublethal doses of azadirachtin on life-table traits and oviposition behaviour of a model organism in toxicological studies, D. melanogaster, were evaluated. The fecundity and oviposition preference of flies surviving to single azadirachtin-treated larvae of parental generation was adversely affected and resulted in the reduction of the number of eggs laid and increased aversion to this compound over two successive generations. In parental generation, early exposure to azadirachtin affects adult's development by reducing the number of organisms, delay larval and pupal development; male biased sex ratio and induced morphological alterations. Moreover, adult's survival of the two generations was significantly decreased as compared to the control. Therefore, Single preimaginal azadirachtin treatment can affect flies population dynamics via transgenerational reductions in survival and reproduction capacity as well as reinforcement of oviposition avoidance which can contribute as repellent strategies in integrated pest management programs. The transgenerational effects observed suggest a possible reduction both in application frequency and total amount of pesticide used, would help in reducing both control costs and possible ecotoxicological risks.
Hsieh, A. L., Zheng, X., Yue, Z., Stine, Z. E., Mancuso, A., Rhoades, S. D., Brooks, R., Weljie, A. M., Eisenman, R. N., Sehgal, A. and Dang, C. V. (2019). Misregulation of Drosophila Myc disrupts circadian behavior and metabolism. Cell Rep 29(7): 1778-1788.e1774. PubMed ID: 31722196
Summary:
Drosophila Myc (dMyc) is highly conserved and functions as a transcription factor similar to mammalian Myc. Previous work has found that oncogenic Myc disrupts the molecular clock in cancer cells. This study demonstrates that misregulation of dMyc expression affects Drosophila circadian behavior. dMyc overexpression results in a high percentage of arrhythmic flies, concomitant with increases in the expression of clock genes cyc, tim, cry, and cwo. Conversely, flies with hypomorphic mutations in dMyc exhibit considerable arrhythmia, which can be rescued by loss of dMnt, a suppressor of dMyc activity. Metabolic profiling of fly heads revealed that loss of dMyc and its overexpression alter steady-state metabolite levels and have opposing effects on histidine, the histamine precursor, which is rescued in dMyc mutants by ablation of dMnt and could contribute to effects of dMyc on locomotor behavior. These results demonstrate a role of dMyc in modulating Drosophila circadian clock, behavior, and metabolism.
Mathejczyk, T. F. and Wernet, M. F. (2019). Heading choices of flying Drosophila under changing angles of polarized light. Sci Rep 9(1): 16773. PubMed ID: 31727972
Summary:
Many navigating insects include the celestial polarization pattern as an additional visual cue to orient their travels. Spontaneous orientation responses of both walking and flying fruit flies (Drosophila melanogaster) to linearly polarized light have previously been demonstrated. Using newly designed modular flight arenas consisting entirely of off-the-shelf parts and 3D-printed components this study presented individual flying flies with a slow and continuous rotational change in the incident angle of linear polarization. Under such open-loop conditions, single flies choose arbitrary headings with respect to the angle of polarized light and show a clear tendency to maintain those chosen headings for several minutes, thereby adjusting their course to the slow rotation of the incident stimulus. Importantly, flies show the tendency to maintain a chosen heading even when two individual test periods under a linearly polarized stimulus are interrupted by an epoch of unpolarized light lasting several minutes. Finally, it was shown that these behavioral responses are wavelength-specific, existing under polarized UV stimulus while being absent under polarized green light. Taken together, these findings provide further evidence supporting Drosophila's abilities to use celestial cues for visually guided navigation and course correction.

Thursday, December 19th - Adult Neural Function

Piggott, B. J., Peters, C. J., He, Y., Huang, X., Younger, S., Jan, L. Y. and Jan, Y. N. (2019). Paralytic, the Drosophila voltage-gated sodium channel, regulates proliferation of neural progenitors. Genes Dev. PubMed ID: 31753914
Summary:
Proliferating cells, typically considered "nonexcitable," nevertheless, exhibit regulation by bioelectric signals. Notably, voltage-gated sodium channels (VGSC) that are crucial for neuronal excitability are also found in progenitors and up-regulated in cancer. This study identified a role for VGSC in proliferation of Drosophila neuroblast (NB) lineages within the central nervous system. Loss of paralytic (para), the sole gene that encodes Drosophila VGSC, reduces neuroblast progeny cell number. The type II neuroblast lineages, featuring a population of transit-amplifying intermediate neural progenitors (INP) similar to that found in the developing human cortex, are particularly sensitive to para manipulation. Following a series of asymmetric divisions, INPs normally exit the cell cycle through a final symmetric division. The data suggest that loss of Para induces apoptosis in this population, whereas overexpression leads to an increase in INPs and overall neuroblast progeny cell numbers. These effects are cell autonomous and depend on Para channel activity. Reduction of Para expression not only affects normal NB development, but also strongly suppresses brain tumor mass, implicating a role for Para in cancer progression. These studies are the first to identify a role for VGSC in neural progenitor proliferation. Elucidating the contribution of VGSC in proliferation will advance understanding of bioelectric signaling within development and disease states.
Liu, Z., Tabuloc, C. A., Xue, Y., Cai, Y., McIntire, P., Niu, Y., Lam, V. H., Chiu, J. C. and Zhang, Y. (2019). Splice variants of DOMINO control Drosophila circadian behavior and pacemaker neuron maintenance. PLoS Genet 15(10): e1008474. PubMed ID: 31658266
Summary:
Circadian clocks control daily rhythms in behavior and physiology. In Drosophila, the small ventral lateral neurons (sLNvs) expressing Pigment Dispersing Factor (PDF) are the master pacemaker neurons generating locomotor rhythms. Despite the importance of sLNvs and PDF in circadian behavior, little is known about factors that control sLNvs maintenance and PDF accumulation. This study identified the Drosophila SWI2/SNF2 protein Domino (Dom) as a key regulator of circadian behavior. Depletion of DOM in circadian neurons eliminates morning anticipatory activity under light dark cycle and impairs behavioral rhythmicity in constant darkness. Interestingly, the two major splice variants of DOM, DOM-A and DOM-B have distinct circadian functions. DOM-A depletion mainly leads to arrhythmic behavior, while DOM-B knockdown lengthens circadian period without affecting the circadian rhythmicity. Both DOM-A and DOM-B bind to the promoter regions of key pacemaker genes period and timeless, and regulate their protein expression. However, this study has identified that only DOM-A is required for the maintenance of sLNvs and transcription of pdf. Lastly, constitutive activation of PDF-receptor signaling rescued the arrhythmia and period lengthening of DOM downregulation. Taken together, these findings reveal that two splice variants of DOM play distinct roles in circadian rhythms through regulating abundance of pacemaker proteins and sLNvs maintenance.
Masuzzo, A., Maniere, G., Viallat-Lieutaud, A., Avazeri, E., Zugasti, O., Grosjean, Y., Kurz, C. L. and Royet, J. (2019). Peptidoglycan-dependent NF-kappaB activation in a small subset of brain octopaminergic neurons controls female oviposition. Elife 8. PubMed ID: 31661076
Summary:
When facing microbes, animals engage in behaviors that lower the impact of the infection. Internal sensing of bacterial peptidoglycan (PGRP-LB) has been shown to reduce Drosophila female oviposition via NF-kappaB pathway activation in some neurons. Although this study showed that the neuromodulator octopamine is implicated, the identity of the involved neurons, as well as the physiological mechanism blocking egg-laying, remained unknown. This study has identified few ventral nerve cord and brain octopaminergic neurons expressing an NF-kappaB pathway component. This study functionally demonstrated that NF-kappaB pathway activation in the brain, but not in the ventral nerve cord octopaminergic neurons, triggers an egg-laying drop in response to infection. Furthermore, it was demonstrated via calcium imaging that the activity of these neurons can be directly modulated by peptidoglycan and that these cells do not control other octopamine-dependent behaviors such as female receptivity. This study shows that by sensing peptidoglycan and hence activating NF-kappaB cascade, a couple of brain neurons modulate a specific octopamine-dependent behavior to adapt female physiology status to their infectious state.
Raccuglia, D., Huang, S., Ender, A., Heim, M. M., Laber, D., Suarez-Grimalt, R., Liotta, A., Sigrist, S. J., Geiger, J. R. P. and Owald, D. (2019). Network-specific synchronization of electrical slow-wave oscillations regulates sleep drive in Drosophila. Curr Biol 29(21): 3611-3621. PubMed ID: 31630955
Summary:
Slow-wave rhythms characteristic of deep sleep oscillate in the delta band (0.5-4 Hz) and can be found across various brain regions in vertebrates. Across phyla, however, an understanding of the mechanisms underlying oscillations and how these link to behavior remains limited. This study discovered compound delta oscillations in the sleep-regulating R5 network of Drosophila. The power of these slow-wave oscillations increases with sleep need and is subject to diurnal variation. Optical multi-unit voltage recordings reveal that single R5 neurons get synchronized by activating circadian input pathways. This synchronization depends on NMDA receptor (NMDAR) coincidence detector function, and an interplay of cholinergic and glutamatergic inputs regulates oscillatory frequency. Genetically targeting the coincidence detector function of NMDARs in R5, and thus the uncovered mechanism underlying synchronization, abolished network-specific compound slow-wave oscillations. It also disrupted sleep and facilitated light-induced wakening, establishing a role for slow-wave oscillations in regulating sleep and sensory gating. It is therefore proposed that the synchronization-based increase in oscillatory power likely represents an evolutionarily conserved, potentially "optimal," strategy for constructing sleep-regulating sensory gates.
Menon, K. P., Kulkarni, V., Takemura, S. Y., Anaya, M. and Zinn, K. (2019). Interactions between Dpr11 and DIP-gamma control selection of amacrine neurons in Drosophila color vision circuits. Elife 8. PubMed ID: 31692445
Summary:
Drosophila R7 UV photoreceptors (PRs) are divided into yellow (y) and pale (p) subtypes. yR7 PRs express the Dpr11 cell surface protein and are presynaptic to Dm8 amacrine neurons (yDm8) that express Dpr11's binding partner DIP-γ, while pR7 PRs synapse onto DIP-γ-negative pDm8. Dpr11 and DIP-g expression patterns define 'yellow' and 'pale' color vision circuits. This study examined Dm8 neurons in these circuits by electron microscopic reconstruction and expansion microscopy. DIP-γ and dpr11 mutations affect the morphologies of yDm8 distal ('home column') dendrites. yDm8 neurons are generated in excess during development and compete for presynaptic yR7 PRs, and interactions between Dpr11 and DIP-γ are required for yDm8 survival. These interactions also allow yDm8 neurons to select yR7 PRs as their appropriate home column partners. yDm8 and pDm8 neurons do not normally compete for survival signals or R7 partners, but can be forced to do so by manipulation of R7 subtype fate.
Ng, R., Salem, S. S., Wu, S. T., Wu, M., Lin, H. H., Shepherd, A. K., Joiner, W. J., Wang, J. W. and Su, C. Y. (2019). Amplification of Drosophila olfactory responses by a DEG/ENaC channel. Neuron. PubMed ID: 31629603
Summary:
Insect olfactory receptors operate as ligand-gated ion channels that directly transduce odor stimuli into electrical signals. However, in the absence of any known intermediate transduction steps, it remains unclear whether and how these ionotropic inputs are amplified in olfactory receptor neurons (ORNs). This study finds that amplification occurs in the Drosophila courtship-promoting ORNs through Pickpocket 25 (PPK25), a member of the degenerin/epithelial sodium channel family (DEG/ENaC). Pharmacological and genetic manipulations indicate that, in Or47b and Ir84a ORNs, PPK25 mediates Ca(2+)-dependent signal amplification via an intracellular calmodulin-binding motif. Additionally, hormonal signaling upregulates PPK25 expression to determine the degree of amplification, with striking effects on male courtship. Together, these findings advance understanding of sensory neurobiology by identifying an amplification mechanism compatible with ionotropic signaling. Moreover, this study offers new insights into DEG/ENaC activation by highlighting a novel means of regulation that is likely conserved across species.

Thursday, December 18th - Stem Cells

Wisidagama, D. R. and Thummel, C. S. (2019). Regulation of Drosophila intestinal stem cell proliferation by enterocyte mitochondrial pyruvate metabolism. G3 (Bethesda). PubMed ID: 31488514
Summary:
Multiple signaling pathways in the adult Drosophila enterocyte sense cellular damage or stress and signal to intestinal stem cells (ISCs) to undergo proliferation and differentiation, thereby maintaining intestinal homeostasis. This study shows that misregulation of mitochondrial pyruvate metabolism in enterocytes can stimulate ISC proliferation and differentiation. These studies focus on the Mitochondrial Pyruvate Carrier (MPC), which is an evolutionarily-conserved protein complex that resides in the inner mitochondrial membrane and transports cytoplasmic pyruvate into the mitochondrial matrix. Loss of MPC function in enterocytes induces Unpaired cytokine expression, which activates the JAK/STAT pathway in ISCs, promoting their proliferation. Upd3 and JNK signaling are required in enterocytes for ISC proliferation, indicating that this reflects a canonical non-cell autonomous damage response. Disruption of lactate dehydrogenase in enterocytes has no effect on ISC proliferation but it suppresses the proliferative response to a loss of enterocyte MPC function, suggesting that lactate contributes to this pathway. These studies define an important role for cellular pyruvate metabolism in differentiated enterocytes to maintain stem cell proliferation rates.
Ranjan, R., Snedeker, J. and Chen, X. (2019). Asymmetric centromeres differentially coordinate with mitotic machinery to ensure biased sister chromatid segregation in germline stem cells. Cell Stem Cell. PubMed ID: 31564548
Summary:
Many stem cells utilize asymmetric cell division (ACD) to produce a self-renewed stem cell and a differentiating daughter cell. How non-genic information could be inherited differentially to establish distinct cell fates is not well understood. This study reports a series of spatiotemporally regulated asymmetric components, which ensure biased sister chromatid attachment and segregation during ACD of Drosophila male germline stem cells (GSCs). First, sister centromeres are differentially enriched with proteins involved in centromere specification and kinetochore function. Second, temporally asymmetric microtubule activities and polarized nuclear envelope breakdown allow for the preferential recognition and attachment of microtubules to asymmetric sister kinetochores and sister centromeres. Abolishment of either the asymmetric sister centromeres or the asymmetric microtubule activities results in randomized sister chromatid segregation. Together, these results provide the cellular basis for partitioning epigenetically distinct sister chromatids during stem cell ACDs, which opens new directions to study these mechanisms in other biological contexts.
Genovese, S., Clement, R., Gaultier, C., Besse, F., Narbonne-Reveau, K., Daian, F., Foppolo, S., Luis, N. M. and Maurange, C. (2019). Coopted temporal patterning governs cellular hierarchy, heterogeneity and metabolism in Drosophila neuroblast tumors. Elife 8. PubMed ID: 31566561
Summary:
It is still unclear what drives progression of childhood tumors. During Drosophila larval development, asymmetrically-dividing neural stem cells, called neuroblasts, progress through an intrinsic temporal patterning program that ensures cessation of divisions before adulthood. Previous work has shown that temporal patterning also delineates an early developmental window during which neuroblasts are susceptible to tumor initiation. Using single-cell transcriptomics, clonal analysis and numerical modeling, this study now identifies a network of twenty larval temporal patterning genes that are redeployed within neuroblast tumors to trigger a robust hierarchical division scheme that perpetuates growth while inducing predictable cell heterogeneity. Along the hierarchy, temporal patterning genes define a differentiation trajectory that regulates glucose metabolism genes to determine the proliferative properties of tumor cells. Thus, partial redeployment of the temporal patterning program encoded in the cell of origin may govern the hierarchy, heterogeneity and growth properties of neural tumors with a developmental origin.
Zhou, Y., Yang, Y., Huang, Y., Wang, H., Wang, S. and Luo, H. (2019). Broad promotes neuroepithelial stem cell differentiation in the Drosophila optic lobe. Genetics. PubMed ID: 31530575
Summary:
Brain development requires the generation of the right number and type of neurons and glial cells at the right time. The Drosophila optic lobe, like mammalian brains, develops from simple neuroepithelia; they first divide symmetrically to expand the progenitor pool and then differentiate into neuroblasts which divide asymmetrically to generate neurons and glial cells. This study investigated the mechanisms that control neuroepithelial growth and differentiation in the optic lobe. The BTB-zinc finger protein Broad, dynamically expressed in the optic lobe neuroepithelia, promotes the transition of neuroepithelial cells to medulla neuroblasts. Loss of Broad function causes neuroepithelial cells to remain highly proliferative and delays neuroepithelial cell differentiation into neuroblasts, which leads to defective lamina and medulla. Conversely, Broad overexpression induces neuroepithelial cells to prematurely transform into medulla neuroblasts. The Ecdysone receptor is required for neuroepithelial maintenance and growth and that Broad expression in neuroepithelial cells is repressed by the Ecdysone receptor. These studies identify Broad as an important cell-intrinsic transcription factor that promotes the neuroepithelial cell to neuroblast transition.
Ma, H., Zhao, H., Liu, F., Zhao, H., Kong, R., Shi, L., Wei, M. and Li, Z. (2019). Heparan sulfate negatively regulates intestinal stem cell proliferation in Drosophila adult midgut. Biol Open 8(10). PubMed ID: 31628141
Summary:
Tissue homeostasis is maintained by differentiated progeny of residential stem cells. Both extrinsic signals and intrinsic factors play critical roles in the proliferation and differentiation of adult intestinal stem cells (ISCs). However, how extrinsic signals are transduced into ISCs still remains unclear. This study finds that heparan sulfate (HS), a class of glycosaminoglycan (GAG) chains, negatively regulates progenitor proliferation and differentiation to maintain midgut homeostasis under physiological conditions. Interestingly, HS depletion in progenitors results in inactivation of Decapentaplegic (Dpp) signaling. Dpp signal inactivation in progenitors resembles HS-deficient intestines. Ectopic Dpp signaling completely rescued the defects caused by HS depletion. Taken together, these data demonstrate that HS is required for Dpp signaling to maintain midgut homeostasis. These results provide insight into the regulatory mechanisms of how extrinsic signals are transduced into stem cells to regulate their proliferation and differentiation.
van den Ameele, J. and Brand, A. (2019). Neural stem cell temporal patterning and brain tumour growth rely on oxidative phosphorylation. Elife 8. PubMed ID: 31513013
Summary:
Translating advances in cancer research to clinical applications requires better insight into the metabolism of normal cells and tumour cells in vivo. Much effort has focused on understanding how glycolysis and oxidative phosphorylation (OxPhos) support proliferation, while their impact on other aspects of development and tumourigenesis remain largely unexplored. This study found that inhibition of OxPhos in neural stem cells (NSCs) or tumours in the Drosophila brain not only decreases proliferation, but also affects many different aspects of stem cell behaviour. In NSCs, OxPhos dysfunction leads to a protracted G1/S-phase and results in delayed temporal patterning and reduced neuronal diversity. As a consequence, NSCs fail to undergo terminal differentiation, leading to prolonged neurogenesis into adulthood. Similarly, in brain tumours inhibition of OxPhos slows proliferation and prevents differentiation, resulting in reduced tumour heterogeneity. Thus, in vivo, highly proliferative stem cells and tumour cells require OxPhos for efficient growth and generation of diversity.

Tuesday, December 17th - Adult neural development and function

Horn, M., Mitesser, O., Hovestadt, T., Yoshii, T., Rieger, D. and Helfrich-Forster, C. (2019). The circadian clock improves fitness in the fruit fly, Drosophila melanogaster. Front Physiol 10: 1374. PubMed ID: 31736790
Summary:
It is assumed that a properly timed circadian clock enhances fitness, but only few studies have truly demonstrated this in animals. Each of the three classical Drosophila period mutants were raised for >50 generations in the laboratory in competition with wildtype flies. The populations were either kept under a conventional 24-h day or under cycles that matched the mutant's natural cycle, i.e., a 19-h day in the case of pers mutants and a 29-h day for perl mutants. The arrhythmic per0 mutants were grown together with wildtype flies under constant light that renders wildtype flies similar arrhythmic as the mutants. In addition, the mutants had to compete with wildtype flies for two summers in two consecutive years under outdoor conditions. Wildtype flies were found to quickly outcompeted the mutant flies under the 24-h laboratory day and under outdoor conditions, but perl mutants persisted and even outnumbered the wildtype flies under the 29-h day in the laboratory. In contrast, pers and per0 mutants did not win against wildtype flies under the 19-h day and constant light, respectively. These results demonstrate that wildtype flies have a clear fitness advantage in terms of fertility and offspring survival over the period mutants and - as revealed for perl mutants - this advantage appears maximal when the endogenous period resonates with the period of the environment. However, the experiments indicate that perl and pers persist at low frequencies in the population even under the 24-h day. This may be a consequence of a certain mating preference of wildtype and heterozygous females for mutant males and time differences in activity patterns between wildtype and mutants.
Hancock, C. E., Bilz, F. and Fiala, A. (2019). In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster. J Vis Exp(152). PubMed ID: 31657798
Summary:
Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented in this study demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells while delivering olfactory stimuli. Additionally, a set-up is demonstrated in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously naive odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. The generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments, has been previously reported. Two-photon microscopy provides a way to spatially resolve fine structures. This is exemplified by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.
Kurmangaliyev, Y. Z., Yoo, J., LoCascio, S. A. and Zipursky, S. L. (2019). Modular transcriptional programs separately define axon and dendrite connectivity. Elife 8. PubMed ID: 31687928
Summary:
Patterns of synaptic connectivity are remarkably precise and complex. Single-cell RNA sequencing has revealed a vast transcriptional diversity of neurons. Nevertheless, a clear logic underlying the transcriptional control of neuronal connectivity has yet to emerge. This study focused on Drosophila T4/T5 neurons, a class of closely related neuronal subtypes with different wiring patterns. Eight subtypes of T4/T5 neurons are defined by combinations of two patterns of dendritic inputs and four patterns of axonal outputs. Single-cell profiling during development revealed distinct transcriptional programs defining each dendrite and axon wiring pattern. These programs were defined by the expression of a few transcription factors and different combinations of cell surface proteins. Gain and loss of function studies provide evidence for independent control of different wiring features. It is proposed that modular transcriptional programs for distinct wiring features are assembled in different combinations to generate diverse patterns of neuronal connectivity.
Li, H., Russo, A. and DiAntonio, A. (2019). SIK3 suppresses neuronal hyperexcitability by regulating the glial capacity to buffer K(+) and water. J Cell Biol. PubMed ID: 31645458
Summary:
Glial regulation of extracellular potassium (K(+)) helps to maintain appropriate levels of neuronal excitability. While channels and transporters mediating K(+) and water transport are known, little is understood about upstream regulatory mechanisms controlling the glial capacity to buffer K(+) and osmotically obliged water. This study identified salt-inducible kinase 3 (SIK3) as the central node in a signal transduction pathway controlling glial K(+) and water homeostasis in Drosophila. Loss of SIK3 leads to dramatic extracellular fluid accumulation in nerves, neuronal hyperexcitability, and seizures. SIK3-dependent phenotypes are exacerbated by K(+) stress. SIK3 promotes the cytosolic localization of HDAC4, thereby relieving inhibition of Mef2-dependent transcription of K(+) and water transport molecules. This transcriptional program controls the glial capacity to regulate K(+) and water homeostasis and modulate neuronal excitability. HDAC4 was identified as a candidate therapeutic target in this pathway, whose inhibition can enhance the K(+) buffering capacity of glia, which may be useful in diseases of dysregulated K(+) homeostasis and hyperexcitability.
Kaur, R., Surala, M., Hoger, S., Grossmann, N., Grimm, A., Timaeus, L., Kallina, W. and Hummel, T. (2019). Pioneer interneurons instruct bilaterality in the Drosophila olfactory sensory map. Sci Adv 5(10): eaaw5537. PubMed ID: 31681838
Summary:
Interhemispheric synaptic connections, a prominent feature in animal nervous systems for the rapid exchange and integration of neuronal information, can appear quite suddenly during brain evolution, raising the question about the underlying developmental mechanism. This study showed in the Drosophila olfactory system that the induction of a bilateral sensory map, an evolutionary novelty in dipteran flies, is mediated by a unique type of commissural pioneer interneurons (cPINs) via the localized activity of the cell adhesion molecule Neuroglian. Differential Neuroglian signaling in cPINs not only prepatterns the olfactory contralateral tracts but also prevents the targeting of ingrowing sensory axons to their ipsilateral synaptic partners. These results identified a sensitive cellular interaction to switch the sequential assembly of diverse neuron types from a unilateral to a bilateral brain circuit organization.
Liu, C., Meng, Z., Wiggin, T. D., Yu, J., Reed, M. L., Guo, F., Zhang, Y., Rosbash, M. and Griffith, L. C. (2019). A serotonin-modulated circuit controls sleep architecture to regulate cognitive function independent of total sleep in Drosophila. Curr Biol 29(21): 3635-3646. PubMed ID: 31668619
Summary:
Both the structure and the amount of sleep are important for brain function. Entry into deep, restorative stages of sleep is time dependent; short sleep bouts selectively eliminate these states. Fragmentation-induced cognitive dysfunction is a feature of many common human sleep pathologies. Whether sleep structure is normally regulated independent of the amount of sleep is unknown. This study shows that in Drosophila melanogaster, activation of a subset of serotonergic neurons fragments sleep without major changes in the total amount of sleep, dramatically reducing long episodes that may correspond to deep sleep states. Disruption of sleep structure results in learning deficits that can be rescued by pharmacologically or genetically consolidating sleep. Two reciprocally connected sets of ellipsoid body neurons were identified that form the heart of a serotonin-modulated circuit that controls sleep architecture. Taken together, these findings define a circuit essential for controlling the structure of sleep independent of its amount.

Monday December 16th - Signaling

Kushnir, T., Bar-Cohen, S., Mooshayef, N., Lange, R., Bar-Sinai, A., Rozen, H., Salzberg, A., Engelberg, D. and Paroush, Z. (2019). An activating mutation in ERK causes hyperplastic tumors in a scribble mutant tissue in Drosophila. Genetics. PubMed ID: 31740452
Summary:
Receptor tyrosine kinase signaling plays prominent roles in tumorigenesis, and activating oncogenic point mutations in the core pathway components Ras, Raf or MEK are prevalent in many types of cancer. Intriguingly, however, analogous oncogenic mutations in the downstream effector kinase ERK have not been described or validated in vivo. To determine if a point mutation could render ERK intrinsically active and oncogenic, this study has assayed in Drosophila the effects of a mutation that confers constitutive activity upon a yeast ERK ortholog, and which has been also identified in a few human tumors. These analyses indicate that a fly ERK ortholog harboring this mutation alone (RolledR80S), and more so in conjunction with the known sevenmaker mutation (RolledR80S+D334N), suppresses multiple phenotypes caused by loss of Ras-Raf-MEK pathway activity, consistent with an intrinsic activity that is independent of upstream signaling. Moreover, expression of RolledR80S and RolledR80S+D334N induces tissue overgrowth in an established Drosophila cancer model. These findings thus demonstrate that activating mutations can bestow ERK with pro-proliferative, tumorigenic capabilities and suggest that Drosophila represents an effective experimental system for determining the oncogenicity of ERK mutants and their response to therapy.
Laffafian, A. and Tepass, U. (2019). Identification of genes required for apical protein trafficking in Drosophila photoreceptor cells. G3 (Bethesda). PubMed ID: 31649044
Summary:
Drosophila melanogaster photoreceptor cells are highly polarized epithelial cells. Their apical membrane is further subdivided into the stalk membrane and the light-sensing rhabdomere. The photo-pigment Rhodopsin1 (Rh1) localizes to the rhabdomere, whereas the apical determinant Crumbs (Crb) is enriched at the stalk membrane. The proteoglycan Eyes shut (Eys) is secreted through the apical membrane into an inter-rhabdomeral space. Rh1, Crb, and Eys are essential for the development of photoreceptor cells, normal vision, and photoreceptor cell survival. Human orthologs of all three proteins have been linked to retinal degenerative diseases. This study describes an RNAi-based screen examining the importance of 237 trafficking-related genes in apical trafficking of Eys, Rh1, and Crb. Twenty-eight genes were found that have an effect on the localization and/or levels of these apical proteins, and several factors were analyzed in more detail. The Arf GEF protein Sec71 was shown to be required for biosynthetic traffic of both apical and basolateral proteins, that the exocyst complex and the microtubule-based motor proteins dynein and kinesin promote the secretion of Eys and Rh1. Syntaxin 7/Avalanche was shown to controls the endocytosis of Rh1, Eys, and Crb.
Kim, M., Du, O. Y., Whitney, R. J., Wilk, R., Hu, J., Krause, H. M., Kavaler, J. and Reed, B. H. (2019). A functional analysis of the Drosophila gene hindsight: Evidence for positive regulation of EGFR signaling. G3 (Bethesda). PubMed ID: 31649045
Summary:
This study has investigated the relationship between the function of the gene hindsight (hnt), the Drosophila homolog of Ras Responsive Element Binding protein-1 (RREB-1), and the EGFR signaling pathway. hnt mutant embryos are defective in EGFR signaling dependent processes, namely chordotonal organ recruitment and oenocyte specification. The temperature sensitive hypomorphic allele hntpebbled is enhanced by the hypomorphic MAPK allele rolled. hnt overexpression results in ectopic DPax2 expression within the embryonic peripheral nervous system, and this effect is EGFR-dependent. Finally, this study shows that the canonical U-shaped embryonic lethal phenotype of hnt, which is associated with premature degeneration of the extraembyonic amnioserosa and a failure in germ band retraction, is rescued by expression of several components of the EGFR signaling pathway (sSpi, Ras85DV12, pntP1) as well as the caspase inhibitor p35. Based on this collection of corroborating evidence, it is suggested that an overarching function of hnt involves the positive regulation of EGFR signaling.
Lattner, J., Leng, W., Knust, E., Brankatschk, M. and Flores-Benitez, D. (2019). Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P2 in Drosophila. Elife 8. PubMed ID: 31697234
Summary:
An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. This study reports that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting betaHeavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P2 and formation of a novel, Moesin- and PI(4,5)P2-enriched apical membrane sac containing microvilli-like structures. These results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.
Kiss, V., Jipa, A., Varga, K., Takats, S., Maruzs, T., Lorincz, P., Simon-Vecsei, Z., Szikora, S., Foldi, I., Bajusz, C., Toth, D., Vilmos, P., Gaspar, I., Ronchi, P., Mihaly, J. and Juhasz, G. (2019). Drosophila Atg9 regulates the actin cytoskeleton via interactions with profilin and Ena. Cell Death Differ. PubMed ID: 31740789
Summary:
Autophagy ensures the turnover of cytoplasm and requires the coordinated action of Atg proteins, some of which also have moonlighting functions in higher eukaryotes. This study shows that the transmembrane protein Atg9 is required for female fertility, and its loss leads to defects in actin cytoskeleton organization in the ovary and enhances filopodia formation in neurons in Drosophila. Atg9 localizes to the plasma membrane anchor points of actin cables and is also important for the integrity of the cortical actin network. Of note, such phenotypes are not seen in other Atg mutants, suggesting that these are independent of autophagy defects. Mechanistically, this study identifies the known actin regulators profilin and Ena/VASP as novel binding partners of Atg9 based on microscopy, biochemical, and genetic interactions. Accordingly, the localization of both profilin and Ena depends on Atg9. Taken together, these data identify a new and unexpected role for Atg9 in actin cytoskeleton regulation.
Kenwrick, K., Mukherjee, A. and Renault, A. D. (2019). Hmgcr promotes a long-range signal to attract germ cells independent of Hedgehog. J Cell Sci. PubMed ID: 31719159
Summary:
During development many cell types migrate along stereotyped routes determined through deployment of cell surface or secreted guidance molecules. While the identity of many of these molecules is known, the distances over which they natively operate can be difficult to determine. This study has quantified the range of an attractive signal for the migration of Drosophila germ cells. Their migration is guided by an attractive signal generated by the expression of genes in the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (Hmgcr) pathway, and by a repulsive signal generated by the expression of Wunens. The attractive signal downstream of Hmgcr is cell-contact independent and acts at long range, the extent of which depends on Hmgcr levels. This range would be sufficient to reach all of the germ cells for their entire migration. Furthermore, Hmgcr-mediated attraction does not require Wunens but can operate simultaneously with Wunen-mediated repulsion. Lastly, several papers posit Hedgehog (Hh) as being the germ cell attractant downstream of hmgcr. This study provides evidence that this is not the case.

Friday, December 13th - Adult Physiology

Dmitrieva, A. S., Ivnitsky, S. B., Maksimova, I. A., Panchenko, P. L., Kachalkin, A. V. and Markov, A. V. (2019). Yeasts affect tolerance of Drosophila melanogaster to food substrate with high NaCl concentration. PLoS One 14(11): e0224811. PubMed ID: 31693706
Summary:
The ability of model animal species, such as Drosophila melanogaster, to adapt quickly to various adverse conditions has been shown in many experimental evolution studies. It is usually assumed by default that such adaptation is due to changes in the gene pool of the studied population of macroorganisms. At the same time, it is known that microbiome can influence biological processes in macroorganisms. In order to assess the possible impact of microbiome on adaptation, an evolutionary experiment was performed in which some D. melanogaster lines were reared on a food substrate with high NaCl concentration while the others were reared on the standard (favourable) substrate. The reproductive efficiency of experimental lines on the high salt substrate was evaluated three years after the experiment started. The tests confirmed that the lines reared on the salty substrate became more tolerant to high NaCl concentration. Moreover, pre-inoculation of the high salt medium with homogenized salt-tolerant flies tended to improve reproductive efficiency of naive flies on this medium (compared to pre-inoculation with homogenized control flies). The analysis of yeast microbiome in fly homogenates revealed significant differences in number and species richness of yeasts between salt-tolerant and control lines. Some individual yeast lines extracted from the salt-tolerant flies improved reproductive efficiency of naive flies on salty substrate (compared to baker's yeast and no yeast controls), whereas the effect of the yeast lines extracted from the control flies tended to be smaller. The yeast Starmerella bacillaris extracted from the salt-tolerant flies showed the strongest positive effect. This yeast is abundant in all salt-tolerant lines, and very rare or absent in all control lines. The results are consistent with the hypothesis that some components of the yeast microbiome of D. melanogaster contribute to to flies' tolerance to food substrate with high NaCl concentration.
Garbuz, D. G., Sverchinsky, D., Davletshin, A., Margulis, B. A., Mitkevich, V., Kulikov, A. M. and Evgen'ev, M. B. (2019). The molecular chaperone Hsp70 from the thermotolerant Diptera species differs from the Drosophila paralog in its thermostability and higher refolding capacity at extreme temperatures. Cell Stress Chaperones. PubMed ID: 31664698
Summary:
Previous work has demonstrated that species of the Stratiomyidae family exhibit higher tolerance to thermal stress in comparison with that of many representatives of Diptera, including Drosophila species. It was hypothesized that species of this group inherited the specific structures of their chaperones from an ancestor of the Stratiomyidae family, and this enabled the descendants to colonize various extreme habitats. To explore this possibility, copies of the Hsp70 genes from Stratiomys singularior, a typical eurythermal species, and Drosophila melanogaster, were cloned and expressed in Escherichia coli for comparison. To investigate the thermal sensitivity of the chaperone function of the inducible 70-kDa heat shock proteins from these species, an in vitro refolding luciferase assay was used. Under conditions of elevated temperature, S. singularior Hsp70 exhibited higher reactivation activity in comparison with D. melanogaster Hsp70 and even human Hsp70. Similarly, S. singularior Hsp70 was significantly more thermostable and showed in vitro refolding activity after preheatment at higher temperatures than D. melanogaster paralog. Thermally induced unfolding experiments using differential scanning calorimetry indicated that Hsp70 from both Diptera species is formed by two domains with different thermal stabilities and that the ATP-binding domain of S. singularior is stable at temperatures 4 degrees higher than that of the D. melanogaster paralog. This study represents the first report that provides direct experimental data indicating that the evolutionary history of a species may result in adaptive changes in the structures of chaperones to enable them to elicit protective functions at extreme environments.
Chambers, M. C., Jacobson, E., Khalil, S. and Lazzaro, B. P. (2019). Consequences of chronic bacterial infection in Drosophila melanogaster. PLoS One 14(10): e0224440. PubMed ID: 31648237
Summary:
Even when successfully surviving an infection, a host often fails to eliminate a pathogen completely and may sustain substantial pathogen burden for the remainder of its life. Using systemic bacterial infection in Drosophila melanogaster, this study characterize chronic infection by three bacterial species from different genera - Providencia rettgeri, Serratia marcescens, and Enterococcus faecalis-following inoculation with a range of doses. To assess the consequences of these chronic infections, the expression was determined of antimicrobial peptide genes, survival of secondary infection, and starvation resistance after one week of infection. While higher infectious doses unsurprisingly lead to higher risk of death, they also result in higher chronic bacterial loads among the survivors for all three infections. All three chronic infections caused significantly elevated expression of antimicrobial peptide genes at one week post-infection and provided generalized protection again secondary bacterial infection. Only P. rettgeri infection significantly influenced resistance to starvation, with persistently infected flies dying more quickly under starvation conditions relative to controls. These results suggest that there is potentially a generalized mechanism of protection against secondary infection, but that other impacts on host physiology may depend on the specific pathogen. It is proposed that chronic infections in D. melanogaster could be a valuable tool for studying tolerance of infection, including impacts on host physiology and behavior.
Koniger, A., Arif, S. and Grath, S. (2019). Three quantitative trait loci explain more than 60% of variation for chill coma recovery time in a natural population of Drosophila ananassae. G3 (Bethesda) 9(11): 3715-3725. PubMed ID: 31690597
Summary:
Ectothermic species such as insects are particularly vulnerable to climatic fluctuations. Nevertheless, many insects that evolved and diversified in the tropics have successfully colonized temperate regions all over the globe. To shed light on the genetic basis of cold tolerance in such species, a quantitative trait locus (QTL) mapping experiment for chill coma recovery time (CCRT) was conducted in Drosophila ananassae, a cosmopolitan species that has expanded its range from tropical to temperate regions. Using a hierarchical mapping approach that combined standard interval mapping and a multiple-QTL model, three QTL were mapped which altogether explained 64% of the phenotypic variance. For two of the identified QTL, evidence was found of epistasis. To narrow down the list of cold tolerance candidate genes, the QTL intervals was cross-referenced with genes that had previously been identified as differentially expressed in response to cold in D. ananassae, and with thermotolerance candidate genes of D. melanogaster. Among the 58 differentially expressed genes that were contained within the QTL, GF15058 showed a significant interaction of the CCRT phenotype and gene expression. Further, the orthologs of four D. melanogaster thermotolerance candidate genes, MtnA, klarsicht, CG5246 (D.ana/GF17132) and CG10383 (D.ana/GF14829) were identified as candidates for cold tolerance in D. ananassae.
Krycer, J. R., Quek, L. E., Francis, D., Fazakerley, D. J., Elkington, S. D., Diaz-Vegas, A., Cooke, K. C., Weiss, F. C., Duan, X., Kurdyukov, S., Zhou, P. X., Tambar, U. K., Hirayama, A., Ikeda, S., Kamei, Y., Soga, T., Cooney, G. J. and James, D. E. (2019). Lactate production is a prioritised feature of adipocyte metabolism. J Biol Chem. PubMed ID: 31690627
Summary:
Adipose tissue is essential for whole-body glucose homeostasis, with a primary role in lipid storage. It has been previously observed that lactate production is also an important metabolic feature of adipocytes, but its relationship to adipose and whole-body glucose disposal remains unclear. Therefore, using a combination of metabolic labeling techniques, this study closely examined lactate production of cultured and primary mammalian adipocytes. Insulin treatment increased glucose uptake and conversion to lactate, with the latter responding more to insulin than did other metabolic fates of glucose. However, lactate production did not just serve as a mechanism to dispose of excess glucose, since it was also observed that lactate production in adipocytes did not solely depend on glucose availability and even occurred independently of glucose metabolism. This suggests that lactate production is prioritized in adipocytes. Furthermore, knocking down lactate dehydrogenase specifically in the fat body of Drosophila flies lowered circulating lactate and improved whole-body glucose disposal. These results emphasize lactate production is an additional metabolic role of adipose tissue beyond lipid storage and release.
Chauhan, V. and Chauhan, A. (2019). Traumatic injury in female Drosophila melanogaster affects the development and induces behavioral abnormalities in the offspring. Behav Brain Funct 15(1): 11. PubMed ID: 31653253
Summary:
Traumatic injury (TI) during pregnancy increases the risk for developing neurological disorders in the infants. These disorders are a major concern for the well-being of children born after TI during pregnancy. TI during pregnancy may result in preterm labor and delivery, abruptio placentae, and/or fetomaternal hemorrhage. Drosophila is a widely used model to study brain and behavioral disorders in humans. This study analyzed the effects of TI to female fruit flies on the development timing of larvae, social interaction and the behavior of offspring flies. TI to the female flies was found to affect the development of larvae and the behavior of offspring flies. There was a significant increase in the length of larvae delivered by traumatically injured maternal flies as compared to larvae from control maternal flies (without TI). The pupae formation from larvae, and the metamorphosis of pupae to the first generation of flies were faster in the TI group than the control group. Negative geotaxis and distance of the fly to its nearest neighbor are parameters of behavioral assessment in fruit flies. Negative geotaxis significantly decreased in the first generation of both male and female flies. The distance between the first generation of flies to its nearest neighbor was shorter in both male and female offspring flies in the TI group as compared to control group flies. These results indicate that TI to the female flies affected the development of larvae and resulted in early delivery, impaired social interaction and behavioral alterations in the offspring.

Thursday, December 13th - Chromatin

Demirdizen, E., Spiller-Becker, M., Fortsch, A., Wilhelm, A., Corless, S., Bade, D., Bergner, A., Hessling, B. and Erhardt, S. (2019). Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex. Nucleic Acids Res. PubMed ID: 31713634
Summary:
Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, different truncation- and localization-deficient CENP-A mutant constructs were used in Drosophila melanogaster cultured cells; the N-terminus of Drosophila melanogaster CENP-A was shown to be required for nuclear localization and protein stability, and CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, these data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.
Umer, Z., Akhtar, J., Khan, M. H. F., Shaheen, N., Haseeb, M. A., Mazhar, K., Mithani, A., Anwar, S. and Tariq, M. (2019). Genome-wide RNAi screen in Drosophila reveals Enok as a novel trithorax group regulator. Epigenetics Chromatin 12(1): 55. PubMed ID: 31547845
Summary:
Polycomb group (PcG) and trithorax group (trxG) proteins contribute to the specialization of cell types by maintaining differential gene expression patterns. This study aimed at discovering novel factors that elicit an anti-silencing effect to facilitate trxG-mediated gene activation. This study has developed a cell-based reporter system and performed a genome-wide RNAi screen to discover novel factors involved in trxG-mediated gene regulation in Drosophila. More than 200 genes were discovered affecting the reporter in a manner similar to trxG genes. From the list of top candidates, (Enoi mushroom), a known histone acetyltransferase, was characterized as an important regulator of trxG in Drosophila. Mutants of enok strongly suppressed extra sex comb phenotype of Pc mutants and enhanced homeotic transformations associated with trx mutations. Enok colocalizes with both TRX and PC at chromatin. Moreover, depletion of Enok specifically resulted in an increased enrichment of PC and consequently silencing of trxG targets. This downregulation of trxG targets was also accompanied by a decreased occupancy of RNA-Pol-II in the gene body, correlating with an increased stalling at the transcription start sites of these genes. It is proposed that Enok facilitates trxG-mediated maintenance of gene activation by specifically counteracting PcG-mediated repression. This ex vivo approach led to identification of new trxG candidate genes that warrant further investigation. Presence of chromatin modifiers as well as known members of trxG and their interactors in the genome-wide RNAi screen validated the reverse genetics approach. Genetic and molecular characterization of Enok revealed a hitherto unknown interplay between Enok and PcG/trxG system. It is concluded that histone acetylation by Enok positively impacts the maintenance of trxG-regulated gene activation by inhibiting PRC1-mediated transcriptional repression.
Viets, K., Sauria, M. E. G., Chernoff, C., Rodriguez Viales, R., Echterling, M., Anderson, C., Tran, S., Dove, A., Goyal, R., Voortman, L., Gordus, A., Furlong, E. E. M., Taylor, J. and Johnston, R. J. (2019). Characterization of button loci that promote homologous chromosome pairing and cell-type-specific interchromosomal gene regulation. Dev Cell. PubMed ID: 31607649
Summary:
Homologous chromosomes colocalize to regulate gene expression in processes including genomic imprinting, X-inactivation, and transvection. In Drosophila, homologous chromosomes pair throughout development, promoting transvection. The "button" model of pairing proposes that specific regions along chromosomes pair with high affinity. This study identified buttons interspersed across the fly genome that pair with their homologous sequences, even when relocated to multiple positions in the genome. A majority of transgenes that span a full topologically associating domain (TAD) function as buttons, but not all buttons contain TADs. Additionally, buttons are enriched for insulator protein clusters. Fragments of buttons do not pair, suggesting that combinations of elements within a button are required for pairing. Pairing is necessary but not sufficient for transvection. Additionally, pairing and transvection are stronger in some cell types than in others, suggesting that pairing strength regulates transvection efficiency between cell types. Thus, buttons pair homologous chromosomes to facilitate cell-type-specific interchromosomal gene regulation.
Kordyukova, M., Sokolova, O., Morgunova, V., Ryazansky, S., Akulenko, N., Glukhov, S. and Kalmykova, A. (2019). Nuclear Ccr4-Not mediates the degradation of telomeric and transposon transcripts at chromatin in the Drosophila germline. Nucleic Acids Res. PubMed ID: 31724732
Summary:
Ccr4-Not is a highly conserved complex involved in cotranscriptional RNA surveillance pathways in yeast. In Drosophila, Ccr4-Not is linked to the translational repression of miRNA targets and the posttranscriptional control of maternal mRNAs during oogenesis and embryonic development. This study describes a new role for the Ccr4-Not complex in nuclear RNA metabolism in the Drosophila germline. Ccr4 depletion results in the accumulation of transposable and telomeric repeat transcripts in the fraction of chromatin-associated RNA; however, it does not affect small RNA levels or the heterochromatin state of the target loci. Nuclear targets of Ccr4 mainly comprise active full-length transposable elements (TEs) and telomeric and subtelomeric repeats. Moreover, Ccr4-Not foci localize at telomeres in a Piwi-dependent manner, suggesting a functional relationship between these pathways. Indeed, this study detected interactions between the components of the Ccr4-Not complex and piRNA machinery, which indicates that these pathways cooperate in the nucleus to recognize and degrade TE transcripts at transcription sites. These data reveal a new layer of transposon control in the germline, which is critical for the maintenance of genome integrity.
Abdusselamoglu, M. D., Landskron, L., Bowman, S. K., Eroglu, E., Burkard, T., Kingston, R. E. and Knoblich, J. A. (2019). Dynamics of activating and repressive histone modifications in Drosophila neural stem cell lineages and brain tumors. Development. PubMed ID: 31748204
Summary:
During central nervous system (CNS) development, spatiotemporal gene expression programs mediate specific lineage decisions to generate neuronal and glial cell types from neural stem cells (NSCs). However, little is known about the epigenetic landscape underlying these highly complex developmental events. This study performed ChIP-seq on distinct subtypes of Drosophila FACS- purified neural stem cells (NSCs) and their differentiated progeny to dissect the epigenetic changes accompanying the major lineage decisions in vivo By analyzing active and repressive histone modifications, this study shows that stem cell identity genes are silenced during differentiation by loss of their activating marks and not via repressive histone modifications. This analysis also uncovers a new set of genes specifically required for altering lineage patterns in type II neuroblasts, one of the two main Drosophila NSC identities. Finally, it was demonstrated that this subtype specification in NBs, unlike NSC differentiation, requires Polycomb-group (PcG)-mediated repression.
Bonnet, J., Lindeboom, R. G. H., Pokrovsky, D., Stricker, G., Celik, M. H., Rupp, R. A. W., Gagneur, J., Vermeulen, M., Imhof, A. and Muller, J. (2019). Quantification of proteins and histone marks in Drosophila embryos reveals stoichiometric relationships impacting chromatin regulation. Dev Cell. PubMed ID: 31630981
Summary:
Gene transcription in eukaryotes is regulated through dynamic interactions of a variety of different proteins with DNA in the context of chromatin. This study used mass spectrometry for absolute quantification of the nuclear proteome and methyl marks on selected lysine residues in histone H3 during two stages of Drosophila embryogenesis. These analyses provide comprehensive information about the absolute copy number of several thousand proteins and reveal unexpected relationships between the abundance of histone-modifying and -binding proteins and the chromatin landscape that they generate and interact with. For some histone modifications, the levels in Drosophila embryos are substantially different from those previously reported in tissue culture cells. Genome-wide profiling of H3K27 methylation during developmental progression and in animals with reduced PRC2 levels illustrates how mass spectrometry can be used for quantitatively describing and comparing chromatin states. Together, these data provide a foundation toward a quantitative understanding of gene regulation in Drosophila.

Wednesday, December 11th - Signaling

Domingos, P. M., Jenny, A., Combie, K. F., Del Alamo, D., Mlodzik, M., Steller, H. and Mollereau, B. (2019). Regulation of Numb during planar cell polarity establishment in the Drosophila eye. Mech Dev: 103583. PubMed ID: 31678471
Summary:
The establishment of planar cell polarity (PCP) in the Drosophila eye requires correct specification of the R3/R4 pair of photoreceptor cells, determined by a Frizzled mediated signaling event that specifies R3 and induces Delta to activate Notch signaling in the neighboring cell, specifying it as R4. This study investigated the role of the Notch signaling negative regulator Numb in the specification of R3/R4 fates and PCP establishment in the Drosophila eye. Numb was observed to be transiently upregulated in R3 at the time of R3/R4 specification. This regulation of Numb levels in developing photoreceptors occurs at the post-transcriptional level and is dependent on Dishevelled, an effector of Frizzled signaling, and Lethal Giant Larva. PCP defects were observed in cells homozygous for numb(15), but these defects were due to a loss of function mutation in fat (fat(Q805)) being present in the numb(15) chromosome. However, mosaic overexpression of Numb in R4 precursors (only) caused PCP defects and numb loss-of-function alleles had a modifying effect on the defects found in a hypomorphic dishevelled mutation. These results suggest that Numb levels are upregulated to reinforce the bias of Notch signaling activation in the R3/R4 pair, two post-mitotic cells that are not specified by asymmetric cell division.
Hopkins, B. R., Sepil, I., Bonham, S., Miller, T., Charles, P. D., Fischer, R., Kessler, B. M., Wilson, C. and Wigby, S. (2019). BMP signaling inhibition in Drosophila secondary cells remodels the seminal proteome and self and rival ejaculate functions. Proc Natl Acad Sci U S A. PubMed ID: 31740617
Summary:
Seminal fluid proteins (SFPs) exert potent effects on male and female fitness. In Drosophila, most SFPs are produced in the accessory glands, which are composed of approximately 1,000 fertility-enhancing "main cells" and approximately 40 more functionally cryptic "secondary cells." Inhibition of bone morphogenetic protein (BMP) signaling in secondary cells suppresses secretion, leading to a unique uncoupling of normal female postmating responses to the ejaculate: refractoriness stimulation is impaired, but offspring production is not. Secondary-cell secretions might regulate more global SFP functions and proteome composition. Secondary-cell-specific BMP signaling inhibition compromises sperm storage and increases female sperm use efficiency. It also impacts second male sperm, tending to slow entry into storage and delay ejection. First male paternity is enhanced, which suggests a constraint on ejaculate evolution whereby high female refractoriness and sperm competitiveness are mutually exclusive. Using quantitative proteomics, this study reveals changes to the seminal proteome that surprisingly encompass alterations to main-cell-derived proteins, indicating important cross-talk between classes of SFP-secreting cells. These results demonstrate that ejaculate composition and function emerge from the integrated action of multiple secretory cell types, suggesting that modification to the cellular make-up of seminal-fluid-producing tissues is an important factor in ejaculate evolution.
Hakim-Mishnaevski, K., Flint-Brodsly, N., Shklyar, B., Levy-Adam, F. and Kurant, E. (2019). Glial phagocytic receptors promote neuronal loss in adult Drosophila brain. Cell Rep 29(6): 1438-1448. PubMed ID: 31693886
Summary:
Glial phagocytosis is critical for the development and maintenance of the CNS in vertebrates and flies and relies on the function of phagocytic receptors to remove apoptotic cells and debris. Glial phagocytic ability declines with age, which correlates with neuronal dysfunction, suggesting that increased glial phagocytosis may prevent neurodegeneration. Contradicting this hypothesis, this study provides experimental evidence showing that an elevated expression of the phagocytic receptors Six-Microns-Under (SIMU) and Draper (Drpr) in adult Drosophila glia leads to a loss of both dopaminergic and GABAergic neurons, accompanied by motor dysfunction and a shortened lifespan. Importantly, this reduction in neuronal number is not linked to neuronal apoptosis, but rather to phosphatidylserine-mediated phagoptosis of live neurons by hyper-phagocytic glia. Altogether, this study reveals that the level of glial phagocytic receptors must be tightly regulated for proper brain function and that neurodegeneration occurs not only by defective, but also excessive glial cell function.
Gonzalez-Morales, N., Xiao, Y. S., Schilling, M. A., Marescal, O., Liao, K. A. and Schock, F. (2019). Myofibril diameter is set by a finely tuned mechanism of protein oligomerization in Drosophila. Elife 8. PubMed ID: 31746737
Summary:
Myofibrils are huge cytoskeletal assemblies embedded in the cytosol of muscle cells. They consist of arrays of sarcomeres, the smallest contractile unit of muscles. Within a muscle type, myofibril diameter is highly invariant and contributes to its physiological properties, yet little is known about the underlying mechanisms setting myofibril diameter. This study shows that the PDZ and LIM domain protein Zasp, a structural component of Z-discs, mediates Z-disc and thereby myofibril growth through protein oligomerization. Oligomerization is induced by an interaction of its ZM domain with LIM domains. Oligomerization is terminated upon upregulation of shorter Zasp isoforms which lack LIM domains at later developmental stages. The balance between these two isoforms, which are call growing and blocking isoforms sets the stereotyped diameter of myofibrils. If blocking isoforms dominate, myofibrils become smaller. If growing isoforms dominate, myofibrils and Z-discs enlarge, eventually resulting in large pathological aggregates that disrupt muscle function.
Hunt, R. J., Granat, L., McElroy, G. S., Ranganathan, R., Chandel, N. S. and Bateman, J. M. (2019). Mitochondrial stress causes neuronal dysfunction via an ATF4-dependent increase in L-2-hydroxyglutarate. J Cell Biol. PubMed ID: 31645461
Summary:
Mitochondrial stress contributes to a range of neurological diseases. Mitonuclear signaling pathways triggered by mitochondrial stress remodel cellular physiology and metabolism. How these signaling mechanisms contribute to neuronal dysfunction and disease is poorly understood. This study finds that mitochondrial stress in neurons activates the transcription factor ATF4 as part of the endoplasmic reticulum unfolded protein response (UPR) in Drosophila. ATF4 activation reprograms nuclear gene expression and contributes to neuronal dysfunction. Mitochondrial stress causes an ATF4-dependent increase in the level of the metabolite L-2-hydroxyglutarate (L-2-HG) in the Drosophila brain. Reducing L-2-HG levels directly, by overexpressing L-2-HG dehydrogenase, improves neurological function. Modulation of L-2-HG levels by mitochondrial stress signaling therefore regulates neuronal function.
Janardan, V., Sharma, S., Basu, U. and Raghu, P. (2019). A genetic screen in Drosophila to identify novel regulation of cell growth by phosphoinositide signaling. G3 (Bethesda). PubMed ID: 31704710
Summary:
Phosphoinositides are lipid signaling molecules that regulate several conserved sub-cellular processes in eukaryotes, including cell growth. Phosphoinositides are generated by the enzymatic activity of highly specific lipid kinases and phosphatases. For example, the lipid PIP3, the Class I PI3 kinase that generates it and the phosphatase PTEN that metabolizes it are all established regulators of growth control in metazoans. To identify additional functions for phosphoinositides in growth control, a genetic screen was performed to identify proteins which when depleted result in altered tissue growth. By using RNA-interference mediated depletion coupled with mosaic analysis in developing eyes, additional candidates were identified and classified in the developing Drosophila melanogaster eye that regulate growth either cell autonomously or via cell-cell interactions. This study reports three genes, Pi3K68D, Vps34 and fwd, that are important for growth regulation and suggest that these are likely to act via cell-cell interactions in the developing eye. These findings define new avenues for the understanding of growth regulation in metazoan tissue development by phosphoinositide metabolizing proteins.

Tuesday, December 10th - Disease Models

Couly, S., Khalil, B., Viguier, V., Roussel, J., Maurice, T. and Lievens, J. C. (2019). Sigma-1 receptor is a key genetic modulator in amyotrophic lateral sclerosis. Hum Mol Genet. PubMed ID: 31696229
Summary:
Sigma-1 receptor (S1R) is an endoplasmic reticulum (ER) chaperone that regulates mitochondrial respiration but also controls cellular defense against ER and oxidative stress. This makes S1R a potential therapeutic target in amyotrophic lateral sclerosis (ALS). Especially, as a missense mutation E102Q in S1R has been reported in few familial ALS cases. However, the pathogenicity of S1RE102Q and the beneficial impact of S1R in the ALS context remain to be demonstrated in vivo. To address this, transgenic Drosophila were generated that express human wild-type S1R or S1RE102Q. Expression of mutant S1R in fly neurons induces abnormal eye morphology and locomotor defects in a dose-dependent manner. This was accompanied by abnormal mitochondrial fragmentation, reduced ATP levels and a higher fatigability at the neuromuscular junction during high energy demand. Overexpressing IP3 receptor or glucose transporter mitigates the S1RE102Q-induced eye phenotype, further highlighting the role of calcium and energy metabolism in its toxicity. More importantly, wild-type S1R rescues locomotor activity and ATP levels of flies expressing the key ALS protein, TDP43. Moreover, overexpressing wild-type S1R enhances resistance of flies to oxidative stress. Therefore, these data provide the first genetic evidence that mutant S1R recapitulates ALS pathology in vivo while increasing S1R confers neuroprotection against TDP43 toxicity.
Ibrahim, R. B., Yeh, S. Y., Lin, K. P., Ricardo, F., Yu, T. Y., Chan, C. C., Tsai, J. W. and Liu, Y. T. (2019). Cellular secretion and cytotoxicity of transthyretin mutant proteins underlie late-onset amyloidosis and neurodegeneration. Cell Mol Life Sci. PubMed ID: 31728576
Summary:
Transthyretin amyloidosis (ATTR) is a progressive life-threatening disease characterized by the deposition of transthyretin (TTR) amyloid fibrils. Several pathogenic variants have been shown to destabilize TTR tetramers, leading to aggregation of misfolded TTR fibrils. However, factors that underlie the differential age of disease onset amongst amyloidogenic TTR variants remain elusive. This study examined the biological properties of various TTR mutations and found that the cellular secretory pattern of the wild-type (WT) TTR was similar to those of the late-onset mutant (Ala97Ser, p. Ala117Ser), stable mutant (Thr119Met, p. Thr139Met), early-onset mutant (Val30Met, p. Val50Met), but not in the unstable mutant (Asp18Gly, p. Asp38Gly). Cytotoxicity assays revealed their toxicities in the order of Val30Met > Ala97Ser > WT > Thr119Met in neuroblastoma cells. Surprisingly, while early-onset amyloidogenic TTR monomers (M-TTRs) are retained by the endoplasmic reticulum quality control (ERQC), late-onset amyloidogenic M-TTRs can be secreted extracellularly. Treatment of thapsigargin (Tg) to activate the unfolded protein response (UPR) alleviates Ala97Ser M-TTR secretion. Interestingly, Ala97Ser TTR overexpression in Drosophila causes late-onset fast neurodegeneration and a relatively short lifespan, recapitulating human disease progression. This study demonstrates that the escape of TTR monomers from the ERQC may underlie late-onset amyloidogenesis in patients and suggests that targeting ERQC could mitigate late-onset ATTR.
Fang, E. F., Hou, Y., Lautrup, S., Jensen, M. B., Yang, B., SenGupta, T., Caponio, D., Khezri, R., Demarest, T. G., Aman, Y., Figueroa, D., Morevati, M., Lee, H. J., Kato, H., Kassahun, H., Lee, J. H., Filippelli, D., Okur, M. N., Mangerich, A., Croteau, D. L., Maezawa, Y., Lyssiotis, C. A., Tao, J., Yokote, K., Rusten, T. E., Mattson, M. P., Jasper, H., Nilsen, H. and Bohr, V. A. (2019). NAD(+) augmentation restores mitophagy and limits accelerated aging in Werner syndrome. Nat Commun 10(1): 5284. PubMed ID: 31754102
Summary:
Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. This study reports impaired mitophagy and depletion of NAD(+), a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD(+) biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD(+) repletion restores NAD(+) metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD(+) repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. These findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD(+) levels counteracts WS phenotypes.
Bervoets, S., Wei, N., Erfurth, M. L., Yusein-Myashkova, S., Ermanoska, B., Mateiu, L., Asselbergh, B., Blocquel, D., Kakad, P., Penserga, T., Thomas, F. P., Guergueltcheva, V., Tournev, I., Godenschwege, T., Jordanova, A. and Yang, X. L. (2019). Transcriptional dysregulation by a nucleus-localized aminoacyl-tRNA synthetase associated with Charcot-Marie-Tooth neuropathy. Nat Commun 10(1): 5045. PubMed ID: 31695036
Summary:
Charcot-Marie-Tooth disease (CMT) is a length-dependent peripheral neuropathy. The aminoacyl-tRNA synthetases constitute the largest protein family implicated in CMT. Aminoacyl-tRNA synthetases are predominantly cytoplasmic, but are also present in the nucleus. This study shows that a nuclear function of tyrosyl-tRNA synthetase (TyrRS) is implicated in a Drosophila model of CMT. CMT-causing mutations in TyrRS induce unique conformational changes, which confer capacity for aberrant interactions with transcriptional regulators in the nucleus, leading to transcription factor E2F1 hyperactivation. Using neuronal tissues, this study revealed a broad transcriptional regulation network associated with wild-type TyrRS expression, which is disturbed when a CMT-mutant is expressed. Pharmacological inhibition of TyrRS nuclear entry with embelin reduces, whereas genetic nuclear exclusion of mutant TyrRS prevents hallmark phenotypes of CMT in the Drosophila model. These data highlight that this translation factor may contribute to transcriptional regulation in neurons, and suggest a therapeutic strategy for CMT.
Gospodaryov, D. V., Strilbytska, O. M., Semaniuk, U. V., Perkhulyn, N. V., Rovenko, B. M., Yurkevych, I. S., Barata, A. G., Dick, T. P., Lushchak, O. V. and Jacobs, H. T. (2019). Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila. Biogerontology. PubMed ID: 31749111
Summary:
Mitochondrial alternative NADH dehydrogenase (aNDH) was found to extend lifespan when expressed in the fruit fly. This study has found that fruit flies expressing aNDH from Ciona intestinalis (NDX) had 17-71% lifespan prolongation on media with different protein-to-carbohydrate ratios except NDX-expressing males that had 19% shorter lifespan than controls on a high protein diet. NDX-expressing flies were more resistant to organic xenobiotics, 2,4-dichlorophenoxyacetic acid and alloxan, and inorganic toxicant potassium iodate, and partially to sodium molybdate treatments. On the other hand, NDX-expressing flies were more sensitive to catechol and sodium chromate. Enzymatic analysis showed that NDX-expressing males had higher glucose 6-phosphate dehydrogenase activity, whilst both sexes showed increased glutathione S-transferase activity.
Imler, E., Pyon, J. S., Kindelay, S., Torvund, M., Zhang, Y. Q., Chandra, S. S. and Zinsmaier, K. E. (2019). A Drosophila model of neuronal ceroid lipofuscinosis CLN4 reveals a hypermorphic gain of function mechanism. Elife 8. PubMed ID: 31663851
Summary:
The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSPalpha. Animal models of CLN4 were developed by expressing CLN4 mutant human CSPalpha (hCSPalpha) in Drosophila neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of hCSPalpha and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSPalpha accumulated abnormally, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructural examination revealed frequent abnormal membrane structures in axons and neuronal somata. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing endogenous wild type (WT) dCSP levels and enhanced by increasing WT levels. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, it is suggested that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.

Monday, December 9th - Adult Development

Shinoda, N., Hanawa, N., Chihara, T., Koto, A. and Miura, M. (2019). Dronc-independent basal executioner caspase activity sustains Drosophila imaginal tissue growth. Proc Natl Acad Sci U S A 116(41): 20539-20544. PubMed ID: 31548372
Summary:
Caspase is best known as an enzyme involved in programmed cell death, which is conserved among multicellular organisms. In addition to its role in cell death, caspase is emerging as an indispensable enzyme in a wide range of cellular functions, which have recently been termed caspase-dependent nonlethal cellular processes (CDPs). This study examined the involvement of cell death signaling in tissue-size determination using Drosophila wing as a model. The Drosophila executioner caspases Dcp-1 and Decay, but not Drice, were found to promote wing growth independently of apoptosis. Most of the reports on CDPs argue the importance of the spatiotemporal regulation of the initiator caspase, Dronc; however, this sublethal caspase function was independent of Dronc, suggesting a more diverse array of CDP regulatory mechanisms. Tagging of TurboID, an improved promiscuous biotin ligase that biotinylates neighboring proteins, to the C terminus of caspases revealed the differences among the neighbors of executioner caspases. Furthermore, this study found that the cleavage of Acinus, a substrate of the executioner caspase, was important in promoting wing growth. These results demonstrate the importance of executioner caspase-mediated basal proteolytic cleavage of substrates in sustaining tissue growth. Given the existence of caspase-like DEVDase activity in a unicellular alga, these results likely highlight the original function of caspase-not cell death, but basal proteolytic cleavages for cell vigor.
Heingard, M., Turetzek, N., Prpic, N. M. and Janssen, R. (2019). FoxB, a new and highly conserved key factor in arthropod dorsal-ventral (DV) limb patterning. Evodevo 10: 28. PubMed ID: 31728178
Summary:
Forkhead box (Fox) transcription factors evolved early in animal evolution and represent important components of conserved gene regulatory networks (GRNs) during animal development. Most of the researches concerning Fox genes, however, are on vertebrates and only a relatively low number of studies investigate Fox gene function in invertebrates. In addition to this shortcoming, the focus of attention is often restricted to a few well-characterized Fox genes such as FoxA (forkhead), FoxC (crocodile) and FoxQ2. Although arthropods represent the largest and most diverse animal group, most other Fox genes have not been investigated in detail, not even in the arthropod model species Drosophila melanogaster. In a general gene expression pattern screen for panarthropod Fox genes including the red flour beetle Tribolium castaneum, the pill millipede Glomeris marginata, the common house spider Parasteatoda tepidariorum, and the velvet worm Euperipatoides kanangrensis, this study identified a Fox gene with a highly conserved expression pattern along the ventral ectoderm of arthropod and onychophoran limbs. Functional investigation of FoxB [i.e., Dmfd4/Dmfd5 (aka fd96Ca/fd96Cb)] in Parasteatoda reveals a hitherto unrecognized important function of FoxB upstream of wingless (wg) and decapentaplegic (dpp) in the GRN orchestrating dorsal-ventral limb patterning.
Tozluoglu, M., Duda, M., Kirkland, N. J., Barrientos, R., Burden, J. J., Munoz, J. J. and Mao, Y. (2019). Planar differential growth rates initiate precise fold positions in complex epithelia. Dev Cell. PubMed ID: 31607650
Summary:
Tissue folding is a fundamental process that shapes epithelia into complex 3D organs. The initial positioning of folds is the foundation for the emergence of correct tissue morphology. Mechanisms forming individual folds have been studied, but the precise positioning of folds in complex, multi-folded epithelia is less well-understood. This paper present a computational model of morphogenesis, encompassing local differential growth and tissue mechanics, to investigate tissue fold positioning. The Drosophila wing disc was used as a model system; there was shown to be spatial-temporal heterogeneity in its planar growth rates. This differential growth, especially at the early stages of development, is the main driver for fold positioning. Increased apical layer stiffness and confinement by the basement membrane drive fold formation but influence positioning to a lesser degree. The model successfully predicts the in vivo morphology of overgrowth clones and wingless mutants via perturbations solely on planar differential growth in silico.
Sauerwald, J., Backer, W., Matzat, T., Schnorrer, F. and Luschnig, S. (2019). Matrix metalloproteinase 1 modulates invasive behavior of tracheal branches during entry into Drosophila flight muscles. Elife 8. PubMed ID: 31577228
Summary:
Tubular networks like the vasculature extend branches throughout animal bodies, but how developing vessels interact with and invade tissues is not well understood. This study investigated the underlying mechanisms using the developing tracheal tube network of Drosophila indirect flight muscles (IFMs) as a model. Live imaging revealed that tracheal sprouts invade IFMs directionally with growth-cone-like structures at branch tips. Ramification inside IFMs proceeds until tracheal branches fill the myotube. However, individual tracheal cells occupy largely separate territories, possibly mediated by cell-cell repulsion. Matrix metalloproteinase 1 (MMP1) is required in tracheal cells for normal invasion speed and for the dynamic organization of growth-cone-like branch tips. MMP1 remodels the CollagenIV-containing matrix around branch tips, which show differential matrix composition with low CollagenIV levels, while Laminin is present along tracheal branches. Thus, tracheal-derived MMP1 sustains branch invasion by modulating the dynamic behavior of sprouting branches as well as properties of the surrounding matrix.
Mumbauer, S., Pascual, J., Kolotuev, I. and Hamaratoglu, F. (2019). Ferritin heavy chain protects the developing wing from reactive oxygen species and ferroptosis. PLoS Genet 15(9): e1008396. PubMed ID: 31568497
Summary:
The interplay between signalling pathways and metabolism is crucial for tissue growth. Yet, it remains poorly understood. The consequences of modulating iron metabolism on the growth of Drosophila imaginal discs were studied. Reducing the levels of the ferritin heavy chain in the larval wing discs was found to lead to drastic growth defects, whereas light chain depletion causes only minor defects. Mutant cell clones for the heavy chain lack the ability to compete against Minute mutant cells. Reactive oxygen species (ROS) accumulate in wing discs with reduced heavy chain levels, causing severe mitochondrial defects and ferroptosis. Preventing ROS accumulation alleviates some of the growth defects. It is proposed that the increased expression of ferritin in hippo mutant cells may protect against ROS accumulation.
Schaub, C., Rose, M. and Frasch, M. (2019). Yorkie and JNK revert syncytial muscles into myoblasts during Org-1-dependent lineage reprogramming. J Cell Biol. PubMed ID: 31591186
Summary:
Lineage reprogramming has received increased research attention since it was demonstrated that lineage-restricted transcription factors can be used in vitro for direct reprogramming. The ventral longitudinal musculature of the adult Drosophila heart has been reported to arise in vivo by direct lineage reprogramming from larval alary muscles, a process that starts with the dedifferentiation and fragmentation of syncytial muscle cells into mononucleate myoblasts and depends on Org-1 (Drosophila Tbx1). This study sheds light on the events occurring downstream of Org-1 in this first step of transdifferentiation and shows that alary muscle lineage-specific activation of Yorkie plays a key role in initiating the dedifferentiation and fragmentation of these muscles. An additional necessary input comes from active dJNK signaling, which contributes to the activation of Yorkie and furthermore activates dJun. The synergistic activities of the Yorkie/Scalloped and dJun/dFos transcriptional activators subsequently initiate alary muscle fragmentation as well as up-regulation of Myc and piwi, both crucial for lineage reprogramming.

Friday, December 6th - Gonads

Biwot, J. C., Zhang, H. B., Liu, C., Qiao, J. X., Yu, X. Q. and Wang, Y. F. (2019). Wolbachia-induced expression of kenny gene in testes affects male fertility in Drosophila melanogaster. Insect Sci. PubMed ID: 31617302
Summary:
Wolbachia are gram-negative endosymbionts that are known to cause embryonic lethality when infected male insects mate with uninfected females or with females carrying a different strain of Wolbachia, a situation characterized as cytoplasmic incompatibility (CI). However, the mechanism of CI is not yet fully understood, although recent studies on Drosophila melanogaster have achieved great progress. This study found that Wolbachia infection caused changes in the expressions of several immunity-related genes, including significant upregulation of kenny (key), in the testes of Drosophila melanogaster. Overexpression of key in fly testes led to a significant decrease in egg hatch rates when these flies mate with wild type females. Wolbachia-infected females could rescue this embryonic lethality. Furthermore, in key overexpressing testes TUNEL signal was significantly stronger than in the control testes, and the level of reactive oxygen species (ROS) was significantly increased. Overexpression of key also resulted in alterations of some other immunity-related gene expressions, including the downregulation of Zn72D. Knockdown of Zn72D in fly testes also led to a significant decrease in egg hatch rates. These results suggest that Wolbachia might induce the defect in male host fertility by immunity-related pathways and thus cause an oxidative damage and cell death in male testes.
Greenblatt, E. J., Obniski, R., Mical, C. and Spradling, A. C. (2019). Prolonged ovarian storage of mature Drosophila oocytes dramatically increases meiotic spindle instability. Elife 8. PubMed ID: 31755866
Summary:
Human oocytes frequently generate aneuploid embryos that subsequently miscarry. In contrast, Drosophila oocytes from outbred laboratory stocks develop fully regardless of maternal age. Since mature Drosophila oocytes are not extensively stored in the ovary under laboratory conditions like they are in the wild, a system was developed to investigate how storage affects oocyte quality. The developmental capacity of stored mature Drosophila oocytes decays in a precise manner over 14 days at 25°C. These oocytes are transcriptionally inactive and persist using ongoing translation of stored mRNAs. Ribosome profiling revealed a progressive 2.3-fold decline in average translational efficiency during storage that correlates with oocyte functional decay. Although normal bipolar meiotic spindles predominate during the first week, oocytes stored for longer periods increasingly show tripolar, monopolar and other spindle defects, and give rise to embryos that fail to develop due to aneuploidy. Thus, meiotic chromosome segregation in mature Drosophila oocytes is uniquely sensitive to prolonged storage. This work suggests the chromosome instability of human embryos could be mitigated by reducing the period of time mature human oocytes are stored in the ovary prior to ovulation.
Wang, Z. H., Liu, Y., Chaitankar, V., Pirooznia, M. and Xu, H. (2019). Electron transport chain biogenesis activated by a JNK-insulin-Myc relay primes mitochondrial inheritance in Drosophila. Elife 8. PubMed ID: 31612862
Summary:
Oogenesis features an enormous increase in mitochondrial mass and mtDNA copy number, which are required to furnish mature eggs with an adequate supply of mitochondria and to curb the transmission of deleterious mtDNA variants. Quiescent in dividing germ cells, mtDNA replication initiates upon oocyte determination in the Drosophila ovary, which necessitates active mitochondrial respiration. However, the underlying mechanism for this dynamic regulation remains unclear. This study shows that an feedforward insulin-Myc loop promotes mitochondrial respiration and biogenesis by boosting the expression of electron transport chain subunits and of factors essential for mtDNA replication and expression, and for the import of mitochondrial proteins. Transient activation of JNK enhances the expression of the insulin receptor and initiates the insulin-Myc signaling loop. This signaling relay promotes mitochondrial biogenesis in the ovary, and thereby plays a role in limiting the transmission of deleterious mtDNA mutations. This study demonstrates cellular mechanisms that couple mitochondrial biogenesis and inheritance with oocyte development.
Vincent, B. J., Rice, G. R., Wong, G. M., Glassford, W. J., Downs, K. I., Shastay, J. L., Charles-Obi, K., Natarajan, M., Gogol, M., Zeitlinger, J. and Rebeiz, M. (2019). An atlas of transcription factors expressed in male pupal terminalia of Drosophila melanogaster. G3 (Bethesda). PubMed ID: 31619460
Summary:
During development, transcription factors and signaling molecules govern gene regulatory networks to direct the formation of unique morphologies. As changes in gene regulatory networks are often implicated in morphological evolution, mapping transcription factor landscapes is important, especially in tissues that undergo rapid evolutionary change. The terminalia (genital and anal structures) of Drosophila melanogaster and its close relatives exhibit dramatic changes in morphology between species. While previous studies have found network components important for patterning the larval genital disc, the networks governing adult structures during pupal development have remained uncharted. RNA-seq was performed in whole Drosophila melanogaster male terminalia followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. The male terminalia are highly patterned during pupal stages and that specific transcription factors mark separate structures and substructures. The results are housed online in a searchable database (https://flyterminalia.pitt.edu) as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia.
Zeledon, C., Sun, X., Plutoni, C. and Emery, G. (2019). The ArfGAP Drongo promotes actomyosin contractility during collective cell migration by releasing Myosin phosphatase from the trailing edge. Cell Rep 28(12): 3238-3248. PubMed ID: 31533044
Summary:
Collective cell migration is involved in various developmental and pathological processes, including the dissemination of various cancer cells. During Drosophila melanogaster oogenesis, a group of cells called border cells migrate collectively toward the oocyte. This study shows that members of the Arf family of small GTPases and some of their regulators are required for normal border cell migration. Notably, it was found that the ArfGAP Drongo and its GTPase-activating function are essential for the initial detachment of the border cell cluster from the basal lamina. Drongo controls the localization of the myosin phosphatase in order to regulate myosin II activity at the back of the cluster. Moreover, toward the class III Arf, Drongo acts antagonistically to the guanine exchange factor Steppke. Overall, this work describes a mechanistic pathway that promotes the local actomyosin contractility necessary for border cell detachment.
Hampoelz, B., Schwarz, A., Ronchi, P., Bragulat-Teixidor, H., Tischer, C., Gaspar, I., Ephrussi, A., Schwab, Y. and Beck, M. (2019). Nuclear pores assemble from Nucleoporin condensates during oogenesis. Cell 179(3): 671-686.e617. PubMed ID: 31626769
Summary:
The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. This study shows that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. This study has delineated a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest.

Thursday, December 5th - Adult neural development and function

Fisher, Y. E., Lu, J., D'Alessandro, I. and Wilson, R. I. (2019). Sensorimotor experience remaps visual input to a heading-direction network. Nature. PubMed ID: 31748749
Summary:
In the Drosophila brain, 'compass' neurons track the orientation of the body and head (the fly's heading) during navigation. In the absence of visual cues, the compass neuron network estimates heading by integrating self-movement signals over time. When a visual cue is present, the estimate of the network is more accurate. Visual inputs to compass neurons are thought to originate from inhibitory neurons called R neurons (also known as ring neurons); the receptive fields of R neurons tile visual space. The axon of each R neuron overlaps with the dendrites of every compass neuron, raising the question of how visual cues are integrated into the compass. Using in vivo whole-cell recordings, this study shows that a visual cue can evoke synaptic inhibition in compass neurons and that R neurons mediate this inhibition. Each compass neuron is inhibited only by specific visual cue positions, indicating that many potential connections from R neurons onto compass neurons are actually weak or silent. It was also shown that the pattern of visually evoked inhibition can reorganize over minutes as the fly explores an altered virtual-reality environment. Using ensemble calcium imaging, it was demonstrated that this reorganization causes persistent changes in the compass coordinate frame. Taken together, these data suggest a model in which correlated pre- and postsynaptic activity triggers associative long-term synaptic depression of visually evoked inhibition in compass neurons. These findings provide evidence for the theoretical proposal that associative plasticity of sensory inputs, when combined with attractor dynamics, can reconcile self-movement information with changing external cues to generate a coherent sense of direction.
Dreyer, A. P., Martin, M. M., Fulgham, C. V., Jabr, D. A., Bai, L., Beshel, J. and Cavanaugh, D. J. (2019). A circadian output center controlling feeding:fasting rhythms in Drosophila. PLoS Genet 15(11): e1008478. PubMed ID: 31693685
Summary:
Circadian rhythms allow animals to coordinate behavioral and physiological processes with respect to one another and to synchronize these processes to external environmental cycles. In most animals, circadian rhythms are produced by core clock neurons in the brain that generate and transmit time-of-day signals to downstream tissues, driving overt rhythms. The neuronal pathways controlling clock outputs, however, are not well understood. Furthermore, it is unclear how the central clock modulates multiple distinct circadian outputs. Identifying the cellular components and neuronal circuitry underlying circadian regulation is increasingly recognized as a critical step in the effort to address health pathologies linked to circadian disruption, including heart disease and metabolic disorders. Building on the conserved components of circadian and metabolic systems in mammals and Drosophila melanogaster, this study used a recently developed feeding monitor to characterize the contribution to circadian feeding rhythms of two key neuronal populations in the Drosophila pars intercerebralis (PI), which is functionally homologous to the mammalian hypothalamus. Thermogenetic manipulations of PI neurons expressing the neuropeptide SIFamide (SIFa) as well as mutations of the SIFa gene degrade feeding:fasting rhythms. In contrast, manipulations of a nearby population of PI neurons that express the Drosophila insulin-like peptides (DILPs) affect total food consumption but leave feeding rhythms intact. The distinct contribution of these two PI cell populations to feeding is accompanied by vastly different neuronal connectivity as determined by trans-Tango synaptic mapping. These results for the first time identify a non-clock cell neuronal population in Drosophila that regulates feeding rhythms and furthermore demonstrate dissociable control of circadian and homeostatic aspects of feeding regulation by molecularly-defined neurons in a putative circadian output hub.
Brunet Avalos, C., Bruggmann, R. and Sprecher, S. G. (2019). Single cell transcriptome atlas of the Drosophila larval brain. Elife 8. PubMed ID: 31746739
Summary:
Cell diversity of the brain and how it is affected by starvation, remains largely unknown. This study introduces a single cell transcriptome atlas of the entire Drosophila first instar larval brain. Cell-type identity was assigned based on known marker genes, distinguishing five major groups: neural progenitors, differentiated neurons, glia, undifferentiated neurons and non-neural cells. All major classes were further subdivided into multiple subtypes, revealing biological features of various cell-types. Transcriptional changes in response to starvation were further assessed at the single-cell level. While after starvation the composition of the brain remains unaffected, transcriptional profile of several cell clusters changed. Intriguingly, different cell-types show very distinct responses to starvation, suggesting the presence of cell-specific programs for nutrition availability. Establishing a single-cell transcriptome atlas of the larval brain provides a powerful tool to explore cell diversity and assess genetic profiles from developmental, functional and behavioral perspectives (Brunet Avalos, 2019).
Chen, Y. D., Park, S. J., Joseph, R. M., Ja, W. W. and Dahanukar, A. A. (2019). Combinatorial pharyngeal taste coding for feeding avoidance in adult Drosophila. Cell Rep 29(4): 961-973.e964. PubMed ID: 31644916
Summary:
Taste drives appropriate food preference and intake. In Drosophila, taste neurons are housed in both external and internal organs, but the latter have been relatively underexplored. This study reports that Poxn mutants with a minimal taste system of pharyngeal neurons can avoid many aversive tastants, including bitter compounds, acid, and salt, suggesting that pharyngeal taste is sufficient for rejecting intake of aversive compounds. Optogenetic activation of selected pharyngeal bitter neurons during feeding events elicits changes in feeding parameters that can suppress intake. Functional dissection experiments indicate that multiple classes of pharyngeal neurons are involved in achieving behavioral avoidance, by virtue of being inhibited or activated by aversive tastants. Tracing second-order pharyngeal circuits reveals two main relay centers for processing pharyngeal taste inputs. Together, these results suggest that the pharynx can control the ingestion of harmful compounds by integrating taste input from different classes of pharyngeal neurons.
Chepurwar, S., Gupta, A., Haddad, R. and Gupta, N. (2019). Sequence-based prediction of olfactory receptor responses. Chem Senses 44(9): 693-703. PubMed ID: 31665762
Summary:
Computational prediction of how strongly an olfactory receptor (OR) responds to various odors can help in bridging the widening gap between the large number of receptors that have been sequenced and the small number of experiments measuring their responses. Previous efforts in this area have predicted the responses of a receptor to some odors, using the known responses of the same receptor to other odors. This study presents a method to predict the responses of a receptor without any known responses by using available data about the responses of other conspecific receptors and their sequences. This method was applied to ORs in insects Drosophila melanogaster (both adult and larva) and Anopheles gambiae and to mouse and human ORs. The predictions are in significant agreement with the experimental measurements. The method also provides clues about the response-determining positions within the receptor sequences.
Dos Santos, J. V., Yu, R. Y., Terceros, A. and Chen, B. E. (2019). FGF receptors are required for proper axonal branch targeting in Drosophila. Mol Brain 12(1): 84. PubMed ID: 31651328
Summary:
Proper axonal branch growth and targeting are essential for establishing a hard-wired neural circuit. This study examined the role of Btl and Htl Fibroblast Growth Factor Receptors (FGFRs) in axonal arbor development using loss of function and overexpression genetic analyses within single neurons. The invariant synaptic connectivity patterns of Drosophila mechanosensory neurons with their innate cleaning reflex responses were used as readouts for errors in synaptic targeting and circuit function. FGFR loss of function resulted in a decrease in axonal branch number and lengths, and overexpression of FGFRs resulted in ectopic branches and increased lengths. FGFR mutants produced stereotyped axonal targeting errors. Both loss of function and overexpression of FGFRs within the mechanosensory neuron decreased the animal's frequency of response to mechanosensory stimulation. The results indicate that FGFRs promote axonal branch growth and proper branch targeting. Disrupting FGFRs results in miswiring and impaired neural circuit function (Dos Santos, 2019).

Wednesday, December 4th - RNAs

Bronkhorst, A. W., Vogels, R., Overheul, G. J., Pennings, B., Gausson-Dorey, V., Miesen, P. and van Rij, R. P. (2019). A DNA virus-encoded immune antagonist fully masks the potent antiviral activity of RNAi in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 31712431
Summary:
Coevolution of viruses and their hosts may lead to viral strategies to avoid, evade, or suppress antiviral immunity. An example is antiviral RNA interference (RNAi) in insects: the host RNAi machinery processes viral double-stranded RNA into small interfering RNAs (siRNAs) to suppress viral replication, whereas insect viruses encode suppressors of RNAi, many of which inhibit viral small interfering RNA (vsiRNA) production. Yet, many studies have analyzed viral RNAi suppressors in heterologous systems, due to the lack of experimental systems to manipulate the viral genome of interest, raising questions about in vivo functions of RNAi suppressors. To address this caveat, an RNAi suppressor-defective mutant was generated of invertebrate iridescent virus 6 (IIV6), a large DNA virus in which the 340R protein has been identified as a suppressor of RNAi. Loss of 340R did not affect vsiRNA production, indicating that 340R binds siRNA duplexes to prevent RNA-induced silencing complex assembly. Indeed, vsiRNAs were not efficiently loaded into Argonaute 2 during wild-type IIV6 infection. Moreover, IIV6 induced a limited set of mature microRNAs in a 340R-dependent manner, most notably miR-305-3p, which is attributed to stabilization of the miR-305-5p:3p duplex by 340R. The IIV6 340R deletion mutant did not have a replication defect in cells, but was strongly attenuated in adult Drosophila. This in vivo replication defect was completely rescued in RNAi mutant flies, indicating that 340R is a bona fide RNAi suppressor, the absence of which uncovers a potent antiviral immune response that suppresses virus accumulation approximately 100-fold. Together, this work indicates that viral RNAi suppressors may completely mask antiviral immunity.
Donelson, N. C., Dixit, R., Pichardo-Casas, I., Chiu, E. Y., Ohman, R. T., Slawson, J. B., Klein, M., Fulga, T. A., Van Vactor, D. and Griffith, L. C. (2019). MicroRNAs regulate multiple aspects of locomotor behavior in Drosophila. G3 (Bethesda). PubMed ID: 31694853
Summary:
Locomotion is an ancient and fundamental output of the nervous system required for animals to perform many other complex behaviors. Although the formation of motor circuits is known to be under developmental control of transcriptional mechanisms that define the fates and connectivity of the many neurons, glia and muscle constituents of these circuits, relatively little is known about the role of post-transcriptional regulation of locomotor behavior. MicroRNAs have emerged as a potentially rich source of modulators for neural development and function. In order to define the microRNAs required for normal locomotion in Drosophila melanogaster, a set of transgenic Gal4-dependent competitive inhibitors (microRNA sponges, or miR-SPs) were used to functionally assess ca. 140 high-confidence Drosophila microRNAs using automated quantitative movement tracking systems followed by multiparametric analysis. Using ubiquitous expression of miR-SP constructs, a large number of microRNAs were identified that modulate aspects of normal baseline adult locomotion. Addition of temperature-dependent Gal80 to identify microRNAs that act during adulthood revealed that the majority of these microRNAs play developmental roles. Comparison of ubiquitous and neural-specific miR-SP expression suggests that most of these microRNAs function within the nervous system. Parallel analyses of spontaneous locomotion in adults and in larvae also reveal that very few of the microRNAs required in the adult overlap with those that control the behavior of larval motor circuits. These screens suggest that a rich regulatory landscape underlies the formation and function of motor circuits and that many of these mechanisms are stage and/or parameter-specific.
Story, B., Ma, X., Ishihara, K., Li, H., Hall, K., Peak, A., Anoja, P., Park, J., Haug, J., Blanchette, M. and Xie, T. (2019). Defining the expression of piRNA and transposable elements in Drosophila ovarian germline stem cells and somatic support cells. Life Sci Alliance 2(5): e201800211. PubMed ID: 31624801
Summary:
Although Piwi-interacting RNAs (piRNAs) are also important for maintaining germline stem cells (GSCs) in the Drosophila ovary by repressing TEs and preventing DNA damage, piRNA expression has not been investigated in GSCs or their early progeny. This study shows that the canonical piRNA clusters are more active in GSCs and their early progeny than late germ cells and also identifies more than 3,000 new piRNA clusters from deep sequencing data. The increase in piRNAs in GSCs and early progeny can be attributed to both canonical and newly identified piRNA clusters. As expected, piRNA clusters in GSCs, but not those in somatic support cells (SCs), exhibit ping-pong signatures. Surprisingly, GSCs and early progeny express more TE transcripts than late germ cells, suggesting that the increase in piRNA levels may be related to the higher levels of TE transcripts in GSCs and early progeny. GSCs also have higher piRNA levels and lower TE levels than SCs. Furthermore, the 3' UTRs of 171 mRNA transcripts may produce sense, antisense, or dual-stranded piRNAs. Finally, it was shown that alternative promoter usage and splicing are frequently used to modulate gene function in GSCs and SCs. Overall, this study has provided important insight into piRNA production and TE repression in GSCs and SCs. The rich information provided by this study will be a beneficial resource to the fields of piRNA biology and germ cell development.
Zhao, K., Cheng, S., Miao, N., Xu, P., Lu, X., Zhang, Y., Wang, M., Ouyang, X., Yuan, X., Liu, W., Lu, X., Zhou, P., Gu, J., Zhang, Y., Qiu, D., Jin, Z., Su, C., Peng, C., Wang, J. H., Dong, M. Q., Wan, Y., Ma, J., Cheng, H., Huang, Y. and Yu, Y. (2019). A Pandas complex adapted for piRNA-guided transcriptional silencing and heterochromatin formation. Nat Cell Biol. PubMed ID: 31570835
Summary:
The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. This study shows that Panoramix functions together with a germline-specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), to suppress transposon expression. The transposon RNA-binding protein dNxf2 is required for animal fertility and Panoramix-mediated silencing. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. The NTF2 domain of dNxf2 competes dNxf1 (TAP) off nucleoporins, a process required for proper RNA export. Thus, dNxf2 functions in a Panoramix-dNxf2-dependent TAP/p15 silencing (Pandas) complex that counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. These findings may have broader implications for understanding how RNA metabolism modulates heterochromatin formation.
Park, J., Zhu, Y., Tao, X., Brazill, J. M., Li, C., Wuchty, S. and Zhai, R. G. (2019). MicroRNA miR-1002 enhances NMNAT-mediated stress response by modulating alternative splicing. iScience 19: 1048-1064. PubMed ID: 31522116
Summary:
Understanding endogenous regulation of stress resistance and homeostasis maintenance is critical to developing neuroprotective therapies. Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a conserved essential enzyme that confers extraordinary protection and stress resistance in many neurodegenerative disease models. Drosophila Nmnat is alternatively spliced to two mRNA variants, RA and RB. RB translates to protein isoform PD with robust protective activity and is upregulated upon stress to confer enhanced neuroprotection. The mechanisms regulating the alternative splicing and stress response of NMNAT remain unclear. A Drosophila microRNA, dme-miR-1002, promotes the splicing of NMNAT pre-mRNA to RB by disrupting a pre-mRNA stem-loop structure. NMNAT pre-mRNA is preferentially spliced to RA in basal conditions, whereas miR-1002 enhances NMNAT PD-mediated stress protection by binding via RISC component Argonaute1 to the pre-mRNA, facilitating the splicing switch to RB. These results outline a new process for microRNAs in regulating alternative splicing and modulating stress resistance.
Bargiela, A., Sabater-Arcis, M., Espinosa-Espinosa, J., Zulaica, M., Lopez de Munain, A. and Artero, R. (2019). Increased Muscleblind levels by chloroquine treatment improve myotonic dystrophy type 1 phenotypes in in vitro and in vivo models. Proc Natl Acad Sci U S A. PubMed ID: 31754023
Summary:
Myotonic dystrophy type 1 (DM1) is a life-threatening and chronically debilitating neuromuscular disease caused by the expansion of a CTG trinucleotide repeat in the 3' UTR of the DMPK gene. The mutant RNA forms insoluble structures capable of sequestering RNA binding proteins of the Muscleblind-like (MBNL) family, which ultimately leads to phenotypes. This work demonstrates that treatment with the antiautophagic drug chloroquine was sufficient to up-regulate MBNL1 and 2 proteins in Drosophila and mouse (HSA(LR)) models and patient-derived myoblasts. Extra Muscleblind was functional at the molecular level and improved splicing events regulated by MBNLs in all disease models. In vivo, chloroquine restored locomotion, rescued average cross-sectional muscle area, and extended median survival in DM1 flies. In HSA(LR) mice, the drug restored muscular strength and histopathology signs and reduced the grade of myotonia. Taken together, these results offer a means to replenish critically low MBNL levels in DM1.

Tuesday, December 3rd - Immune response

Lin, Y. H., Maaroufi, H. O., Ibrahim, E., Kucerova, L. and Zurovec, M. (2019). Expression of human mutant Huntingtin protein in Drosophila hemocytes impairs immune responses. Front Immunol 10: 2405. PubMed ID: 31681295
Summary:
The pathogenic effect of mutant HTT (mHTT) which causes Huntington disease (HD) are not restricted to nervous system. Such phenotypes include aberrant immune responses observed in the HD models. However, it is still unclear how this immune dysregulation influences the innate immune response against pathogenic infection. This study used transgenic Drosophila melanogaster expressing mutant HTT protein (mHTT) with hemocyte-specific drivers and examined the immune responses and hemocyte function. mHTT expression in the hemocytes did not affect fly viability, but the numbers of circulating hemocytes were significantly decreased. Consequently, it was observed that the expression of mHTT in the hemocytes compromised the immune responses including clot formation and encapsulation which lead to the increased susceptibility to entomopathogenic nematode and parasitoid wasp infections. In addition, mHTT expression in Drosophila macrophage-like S2 cells in vitro reduced ATP levels, phagocytic activity and the induction of antimicrobial peptides. Further effects observed in mHTT-expressing cells included the altered production of cytokines and activation of JAK/STAT signaling. The present study shows that the expression of mHTT in Drosophila hemocytes causes deficient cellular and humoral immune responses against invading pathogens. These findings provide the insight into the pathogenic effects of mHTT in the immune cells.
Liao, J. F., Wu, C. P., Tang, C. K., Tsai, C. W., Rouhova, L. and Wu, Y. L. (2019). Identification of regulatory host genes involved in sigma virus replication using RNAi knockdown in Drosophila. Insects 10(10). PubMed ID: 31614679
Summary:
The Drosophila melanogaster sigma virus, a member of the Rhabdoviridae family, specifically propagates itself in D. melanogaster. Sigma-virus-infected Drosophila may suffer from irreversible paralysis when exposed to a high CO2 concentration, but generally, no other symptoms are reported. The present study aimed to identify host genes associated with sigma virus replication. The results showed that the genome copy number of the sigma virus increased after knocking down the immune pathway genes domeless and PGRP-LC in Drosophila S2 cells. The knocking down of domeless and PGRP-LC significantly up-regulated the expression of the L gene, one of six genes encoded by the virus. It is proposed that the immune pathways respond to sigma virus infection by altering L expression, hence suppressing viral replication. This effect was further tested in vivo, when D. melanogaster individuals injected with dsdome and dsPGRP-LC showed not only an increase in sigma virus copy number, but also a reduced survival rate when treated with CO2. This study proved that host immunity influences viral replication, even in persistent infection. It is proposed that the immunity system of D. melanogaster regulates the replication of the sigma virus by affecting the L gene expression. Studies have shown minimal host-virus interaction in persistent infection. However, this study demonstrated that the immunity continued to affect viral replication even in persistent infection because knocking down the key components of the immune process disabled the relevant immune controls and facilitated viral expression and replication.
Bozler, J., Kacsoh, B. Z. and Bosco, G. (2019). Maternal priming of offspring immune system in Drosophila. G3 (Bethesda). PubMed ID: 31685524
Summary:
Immune priming occurs when a past infection experience leads to a more effective immune response upon a secondary exposure to the infection or pathogen. In some instances, parents are able to transmit immune priming to their offspring, creating a subsequent generation with a superior immune capability, through processes that are not yet fully understood. Using a parasitoid wasp, which infects larval stages of Drosophila melanogaster, this study describes an example of an intergenerational inheritance of immune priming. This phenomenon is anticipatory in nature and does not rely on parental infection, but rather, when adult fruit flies are cohabitated with a parasitic wasp, they produce offspring that are more capable of mounting a successful immune response against a parasitic macro-infection. This increase in offspring survival correlates with a more rapid induction of lamellocytes, a specialized immune cell. RNA-sequencing of the female germline identifies several differentially expressed genes following wasp exposure, including the peptiodoglycan recognition protein-LB (PGRP-LB). Genetic manipulation of maternal PGRP-LB identifies this gene as a key element in this intergenerational phenotype.
Chmiel, J. A., Daisley, B. A., Burton, J. P. and Reid, G. (2019). Deleterious effects of Neonicotinoid pesticides on Drosophila melanogaster immune pathways. MBio 10(5). PubMed ID: 31575764
Summary:
dual oxidase (Duox) pathway is a critical component of the innate immune response, which functions to impede infection and maintain homeostatic regulation of the gut microbiota. Despite the importance of this pathway in gut immunity, the consequences of neonicotinoid exposure on Duox signaling have yet to be studied. This study used a Drosophila melanogaster model to investigate the hypothesis that imidacloprid (a common neonicotinoid) can affect the Duox pathway. The results demonstrated that exposure to sublethal imidacloprid reduced H2O2 production by inhibiting transcription of the Duox gene. Furthermore, the reduction in Duox expression was found to be a result of imidacloprid interacting with the midgut portion of the immune deficiency pathway. This impairment led to a loss of microbial regulation, as exemplified by a compositional shift and increased total abundance of Lactobacillus and Acetobacter spp. (dominant microbiota members) found in the gut. In addition, this study demonstrated that certain probiotic lactobacilli could ameliorate Duox pathway impairment caused by imidacloprid, but this effect was not directly dependent on the Duox pathway itself. This study is the first to demonstrate the deleterious effects that neonicotinoids can have on Duox-mediated generation of H2O2 and highlights a novel coordination between two important innate immune pathways present in insects.
Houtz, P., Bonfini, A., Bing, X. and Buchon, N. (2019). Recruitment of adult precursor cells underlies limited repair of the infected larval midgut in Drosophila. Cell Host Microbe 26(3): 412-425. PubMed ID: 31492656
Summary:
Surviving infection requires immune and repair mechanisms. Developing organisms face the additional challenge of integrating these mechanisms with tightly controlled developmental processes. The larval Drosophila midgut lacks dedicated intestinal stem cells. Upon infection, larvae perform limited repair using adult midgut precursors (AMPs). AMPs differentiate in response to damage to generate new enterocytes, transiently depleting their pool. Developmental delay allows for AMP reconstitution, ensuring the completion of metamorphosis. Notch signaling is required for the differentiation of AMPs into the encasing, niche-like peripheral cells (PCs), but not to differentiate PCs into enterocytes. Dpp (TGF-beta) signaling is sufficient, but not necessary, to induce PC differentiation into enterocytes. Infection-induced JAK-STAT pathway is both required and sufficient for differentiation of AMPs and PCs into new enterocytes. Altogether, this work highlights the constraints imposed by development on an organism's response to infection and demonstrates the transient use of adult precursors for tissue repair.
Troha, K., Nagy, P., Pivovar, A., Lazzaro, B. P., Hartley, P. S. and Buchon, N. (2019). Nephrocytes remove microbiota-derived peptidoglycan from systemic circulation to maintain immune homeostasis. Immunity 51(4): 625-637.e623. PubMed ID: 31564469
Summary:
Preventing aberrant immune responses against the microbiota is essential for the health of the host. Microbiota-shed pathogen-associated molecular patterns translocate from the gut lumen into systemic circulation. This study examined the role of hemolymph (insect blood) filtration in regulating systemic responses to microbiota-derived peptidoglycan. Drosophila deficient for the transcription factor Klf15 (Klf15(NN)) are viable but lack nephrocytes-cells structurally and functionally homologous to the glomerular podocytes of the kidney. Klf15(NN) flies were more resistant to infection than wild-type (WT) counterparts but exhibited a shortened lifespan. This was associated with constitutive Toll pathway activation triggered by excess peptidoglycan circulating in Klf15(NN) flies. In WT flies, peptidoglycan was removed from systemic circulation by nephrocytes through endocytosis and subsequent lysosomal degradation. Thus, renal filtration of microbiota-derived peptidoglycan maintains immune homeostasis in Drosophila, a function likely conserved in mammals and potentially relevant to the chronic immune activation seen in settings of impaired blood filtration.

Monday, December 2nd - Signaling

Stinchfield, M. J., Miyazawa, K. and Newfeld, S. J. (2019). Transgenic analyses in Drosophila reveal that mCORL1 is functionally distinct from mCORL2 and dCORL. G3 (Bethesda). PubMed ID: 31530634
Summary:
Uncovering how new members of multigene families acquire new functions is an important topic in evolutionary and developmental genetics. CORL proteins (SKOR in mice, Fussel in humans and fussel in Drosophila) are a family of CNS specific proteins related to mammalian Sno/Ski oncogenes. Drosophila CORL (dCORL/Fussel) participates in TGF-beta and insulin signaling during development and in adult homeostasis but roles for the two mouse CORL proteins (mCORL) are essentially unknown. A series of studies were conducted to test the hypothesis based on previous results that mCORL1 is more similar to dCORL than mCORL2. Neither an updated alignment nor ectopic expression in adult wings were able to distinguish mCORL1 or mCORL2 from dCORL. Transgene experiments employing a dCORL endogenous function in mushroom body neurons showed that mCORL1 is distinct from mCORL2 and dCORL. mCORL1 and mCORL2 are also distinct in biochemical assays of Smad-binding and BMP signaling. Taken together, the data suggests testable new hypotheses for mCORL2 function in mammalian TGF-beta and insulin signaling based on known roles for dCORL. Overall, the study reiterates the value of transgenic methods in Drosophila to provide new information on multigene family evolution and the function of family members in other species.
Barata, A. G. and Dick, T. P. (2019). A role for peroxiredoxins in H2O2- and MEKK-dependent activation of the p38 signaling pathway. Redox Biol 28: 101340. PubMed ID: 31629169
Summary:
The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in the cellular response to various stresses and its deregulation accompanies pathological conditions such as cancer and chronic inflammation. Hydrogen peroxide (H2O2) is a well-established activator of the p38 MAPK signaling pathway. However, the mechanisms of H2O2-induced p38 activation are not yet fully understood. In Drosophila cells, H2O2-induced activation of p38 was found to depend on the MAPK kinase kinase (MAP3K) Mekk1. In line with the emerging role of peroxiredoxins as H2O2 sensors and signal transmitters, an H2O2-dependent interaction was observed between Mekk1 and the cytosolic peroxiredoxin of Drosophila, Jafrac1. In human cells, MEKK4 (the homologue of Mekk1) and peroxiredoxin-2 (Prx2) interact in a similar manner, suggesting an evolutionarily conserved mechanism. In both organisms, H2O2 induces transient disulfide-linked conjugates between the MAP3K and a typical 2-Cys peroxiredoxin. It is proposed that these conjugates represent the relaying of oxidative equivalents from H2O2 to the MAP3K and that the oxidation of Mekk1/MEKK4 leads to the downstream activation of p38 MAPK. Indeed, the depletion of cytosolic 2-Cys peroxiredoxins in human cells diminished H2O2-induced activation of p38 MAPK.
Patel, P. H., Penalva, C., Kardorff, M., Roca, M., Pavlovic, B., Thiel, A., Teleman, A. A. and Edgar, B. A. (2019). Damage sensing by a Nox-Ask1-MKK3-p38 signaling pathway mediates regeneration in the adult Drosophila midgut. Nat Commun 10(1): 4365. PubMed ID: 31554796
Summary:
Epithelia are exposed to diverse types of stress and damage from pathogens and the environment, and respond by regenerating. Yet, the proximal mechanisms that sense epithelial damage remain poorly understood. This paper reports that p38 signaling is activated in adult Drosophila midgut enterocytes in response to diverse stresses including pathogenic bacterial infection and chemical and mechanical insult. Two upstream kinases, Ask1 and Licorne (MKK3), are required for p38 activation following infection, oxidative stress, detergent exposure and wounding. Ask1-p38 signaling in enterocytes is required upon infection to promote full intestinal stem cell (ISC) activation and regeneration, partly through Upd3/Jak-Stat signaling. Furthermore, reactive oxygen species (ROS) produced by the NADPH oxidase Nox in enterocytes, are required for p38 activation in enterocytes following infection or wounding, and for ISC activation upon infection or detergent exposure. It is proposed that Nox-ROS-Ask1-MKK3-p38 signaling in enterocytes integrates multiple different stresses to induce regeneration.
Wilson, C., Kavaler, J. and Ahmad, S. T. (2019). Expression of a human variant of CHMP2B linked to neurodegeneration in Drosophila external sensory organs leads to cell fate transformations associated with increased Notch activity. Dev Neurobiol. PubMed ID: 31587468
Summary:
Proper function of cell signaling pathways is dependent upon regulated membrane trafficking events that lead to the endocytosis, recycling, and degradation of cell surface receptors. The endosomal complexes required for transport (ESCRT) genes play a critical role in the sorting of ubiquinated cell surface proteins. CHMP2B(Intron5) , a truncated form of a human ESCRT-III protein, was discovered in a Danish family afflicted by a hereditary form of frontotemporal dementia (FTD). Although the mechanism by which the CHMP2B mutation in this family causes FTD is unknown, the resulting protein has been shown to disrupt normal endosomal-lysosomal pathway function and leads to aberrant regulation of signaling pathways. This study has misexpressed CHMP2B(Intron5) in the developing Drosophila external sensory (ES) organ lineage; it was shown to be capable of altering cell fates. Each of the cell fate transformations seen is compatible with an increase in Notch signaling. Furthermore, this interpretation is supported by evidence that expression of CHMP2B(Intron5) in the notum environment is capable of raising the levels of Notch signaling. As such, these results add to a growing body of evidence that CHMP2B(Intron5) can act rapidly to disrupt normal cellular function via the misregulation of critical cell surface receptor function.
Zhou, J., Xu, L., Duan, X., Liu, W., Zhao, X., Wang, X., Shang, W., Fang, X., Yang, H., Jia, L., Bai, J., Zhao, J., Wang, L. and Tong, C. (2019). Large-scale RNAi screen identified Dhpr as a regulator of mitochondrial morphology and tissue homeostasis. Sci Adv 5(9): eaax0365. PubMed ID: 31555733
Summary:
Mitochondria are highly dynamic organelles. Through a large-scale in vivo RNA interference (RNAi) screen that covered around a quarter of the Drosophila melanogaster genes (4000 genes), this study has identified 578 genes whose knockdown led to aberrant shapes or distributions of mitochondria. The complex analysis revealed that knockdown of the subunits of proteasomes, spliceosomes, and the electron transport chain complexes could severely affect mitochondrial morphology. The loss of Dhpr, a gene encoding an enzyme catalyzing tetrahydrobiopterin regeneration, leads to a reduction in the numbers of tyrosine hydroxylase neurons, shorter lifespan, and gradual loss of muscle integrity and climbing ability. The affected mitochondria in Dhpr mutants are swollen and have fewer cristae, probably due to lower levels of Drp1 S-nitrosylation. Overexpression of Drp1, but not of S-nitrosylation-defective Drp1, rescued Dhpr RNAi-induced mitochondrial defects. It is proposed that Dhpr regulates mitochondrial morphology and tissue homeostasis by modulating S-nitrosylation of Drp1.
Ahlers, L. R. H., Trammell, C. E., Carrell, G. F., Mackinnon, S., Torrevillas, B. K., Chow, C. Y., Luckhart, S. and Goodman, A. G. (2019). Insulin potentiates JAK/STAT signaling to broadly inhibit flavivirus replication in insect vectors. Cell Rep 29(7): 1946-1960.e1945. PubMed ID: 31722209
Summary:
The World Health Organization estimates that more than half of the world's population is at risk for vector-borne diseases, including arboviruses. Because many arboviruses are mosquito borne, investigation of the insect immune response will help identify targets to reduce the spread of arboviruses. This study used a genetic screening approach to identify an insulin-like receptor as a component of the immune response to arboviral infection. Vertebrate insulin reduces West Nile virus (WNV) replication in Drosophila melanogaster as well as WNV, Zika, and dengue virus titers in mosquito cells. Mechanistically, insulin signaling was shown to activate the JAK/STAT, but not RNAi, pathway via ERK to control infection in Drosophila cells and Culex mosquitoes through an integrated immune response. Finally, insulin priming of adult female Culex mosquitoes through a blood meal reduces WNV infection, demonstrating an essential role for insulin signaling in insect antiviral responses to human pathogens.
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