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Thursday, October 31st, 2019 - Signaling

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Balasubramanian, V. and Srinivasan, B. (2019). Genetic analyses uncover pleiotropic compensatory roles for Drosophila Nucleobindin-1 in inositol trisphosphate-mediated intracellular calcium homeostasis. Genome. PubMed ID: 31557446
Summary:
Nucleobindin-1 is an EF-hand calcium-binding protein with a distinctive profile, predominantly localized to the Golgi in insect and wide-ranging vertebrate cell types, alike. Its putative involvements in intracellular calcium (Ca2+) homeostasis have however never been phenotypically characterized in any model organism. This study has analyzed an adult-viable mutant that completely disrupts the G protein alpha-subunit Binding and Activating (GBA) motif of Drosophila Nucleobindin-1 (dmNUCB1). Such disruption does not manifest any obvious fitness-related, morphological / developmental or behavioral abnormalities. A single copy of this mutation or the knockdown of dmnucb1 in restricted sets of cells, however variously rescues pleiotropic mutant phenotypes arising from impaired Inositol 1,4,5-trisphosphate receptor (IP3R) activity, in turn depleting cytoplasmic Ca2+ levels across diverse tissue types. Additionally, altered dmNUCB1 expression or function considerably reverses lifespan and mobility improvements effected by IP3R mutants, in a Drosophila model of Amyotrophic Lateral Sclerosis. Homology modeling-based analyses further predict a high degree of conformational conservation in Drosophila, of biochemically validated structural determinants in the GBA motif that specify in vertebrates, the unconventional Ca2+-regulated interaction of NUCB1 with Galphai subunits. The broad implications of these findings are hypothetically discussed, regarding potential roles for NUCB1 in GBA-mediated, Golgi-associated Ca2+ signaling, in health and disease.
Borreguero-Munoz, N., Fletcher, G. C., Aguilar-Aragon, M., Elbediwy, A., Vincent-Mistiaen, Z. I. and Thompson, B. J. (2019). The Hippo pathway integrates PI3K-Akt signals with mechanical and polarity cues to control tissue growth. PLoS Biol 17(10): e3000509. PubMed ID: 31613895
Summary:
The Hippo signalling pathway restricts cell proliferation in animal tissues by inhibiting Yes-associated protein (YAP or YAP1) and Transcriptional Activator with a PDZ domain (TAZ), coactivators of the Scalloped (Sd or TEAD) DNA-binding transcription factor. Drosophila has a single YAP/TAZ homolog named Yorkie (Yki) that is regulated by Hippo pathway signalling in response to epithelial polarity and tissue mechanics during development. This study shows that Yki translocates to the nucleus to drive Sd-mediated cell proliferation in the ovarian follicle cell epithelium in response to mechanical stretching caused by the growth of the germline. Importantly, mechanically induced Yki nuclear localisation also requires nutritionally induced insulin/insulin-like growth factor 1 (IGF-1) signalling (IIS) via phosphatidyl inositol-3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1 or PDPK1), and protein kinase B (Akt or PKB) in the follicular epithelium. Similar results were obtained in the developing Drosophila wing, where Yki becomes nuclear in the mechanically stretched cells of the wing pouch during larval feeding, which induces IIS, but translocates to the cytoplasm upon cessation of feeding in the third instar stage. Inactivating Akt prevents nuclear Yki localisation in the wing disc, while ectopic activation of the insulin receptor, PI3K, or Akt/PKB is sufficient to maintain nuclear Yki in mechanically stimulated cells of the wing pouch even after feeding ceases. Finally, IIS also promotes YAP nuclear localisation in response to mechanical cues in mammalian skin epithelia. Thus, the Hippo pathway has a physiological function as an integrator of epithelial cell polarity, tissue mechanics, and nutritional cues to control cell proliferation and tissue growth in both Drosophila and mammals.
Benton, M. A., Frey, N., Nunes da Fonseca, R., von Levetzow, C., Stappert, D., Hakeemi, M. S., Conrads, K. H., Pechmann, M., Panfilio, K. A., Lynch, J. A. and Roth, S. (2019). Fog signaling has diverse roles in epithelial morphogenesis in insects. Elife 8. PubMed ID: 31573513
Summary:
The Drosophila Fog pathway represents one of the best-understood signaling cascades controlling epithelial morphogenesis. During gastrulation, Fog induces apical cell constrictions that drive the invagination of mesoderm and posterior gut primordia. The cellular mechanisms underlying primordia internalization vary greatly among insects and recent work has suggested that Fog signaling is specific to the fast mode of gastrulation found in some flies. On the contrary, this study shows in the beetle Tribolium, whose development is broadly representative for insects, that Fog has multiple morphogenetic functions. It modulates mesoderm internalization and controls a massive posterior infolding involved in gut and extraembryonic development. In addition, Fog signaling affects blastoderm cellularization, primordial germ cell positioning, and cuboidal-to-squamous cell shape transitions in the extraembryonic serosa. Comparative analyses with two other distantly related insect species reveals that Fog's role during cellularization is widely conserved and therefore might represent the ancestral function of the pathway.
Fereres, S., Hatori, R., Hatori, M. and Kornberg, T. B. (2019). Cytoneme-mediated signaling essential for tumorigenesis. PLoS Genet 15(9): e1008415. PubMed ID: 31568500
Summary:
Communication between neoplastic cells and cells of their microenvironment is critical to cancer progression. To investigate the role of cytoneme-mediated signaling as a mechanism for distributing growth factor signaling proteins between tumor and tumor-associated cells, EGFR and RET Drosophila tumor models were analyzed, and several genetic loss-of-function conditions were tested that impair cytoneme-mediated signaling. Neuroglian, capricious, cIrk2, SCAR, and diaphanous are genes that cytonemes require during normal development. Neuroglian and Capricious are cell adhesion proteins, Irk2 is a potassium channel, and SCAR and Diaphanous are actin-binding proteins, and the only process to which they are known to contribute jointly is cytoneme-mediated signaling. It was observed that diminished function of any one of these genes suppressed tumor growth and increased organism survival. It was also noted that EGFR-expressing tumor discs have abnormally extensive tracheation (respiratory tubes) and ectopically express Branchless (Bnl, a FGF) and FGFR. Bnl is a known inducer of tracheation that signals by a cytoneme-mediated process in other contexts, and it was determined that exogenous over-expression of dominant negative FGFR suppressed tumor growth. These results are consistent with the idea that cytonemes move signaling proteins between tumor and stromal cells and that cytoneme-mediated signaling is required for tumor growth and malignancy.
Chen, H., Ronau, J. A., Beckmann, J. F. and Hochstrasser, M. (2019). A Wolbachia nuclease and its binding partner provide a distinct mechanism for cytoplasmic incompatibility. Proc Natl Acad Sci U S A. PubMed ID: 31615889
Summary:
Wolbachia are endosymbiotic bacteria that infect nearly half of all arthropod species. This pandemic is due in part to their ability to increase their transmission through the female germline, most commonly by a mechanism called cytoplasmic incompatibility (CI). The Wolbachia cid operon, encoding 2 proteins, CidA and CidB, the latter a deubiquitylating enzyme (DUB), recapitulates CI in transgenic Drosophila melanogaster. However, some CI-inducing Wolbachia strains lack a DUB-encoding cid operon; it was therefore proposed that the related cin operon codes for an alternative CI system. This study shows that the Wolbachia cin operon encodes a nuclease, CinB, and a second protein, CinA, that tightly binds CinB. Recombinant CinB has nuclease activity against both single-stranded and double-stranded DNA but not RNA under the conditions tested. Expression of the cid operon in transgenic male flies induces male sterility and embryonic defects typical of CI. Importantly, transgenic CinA can rescue defects in egg-hatch rates when expressed in females. Expression of CinA also rescues CinB-induced growth defects in yeast. CinB has 2 PD-(D/E)xK nuclease domains, and both are required for nuclease activity and for toxicity in yeast and flies. These data suggest a distinct mechanism for CI involving a nuclease toxin and highlight the central role of toxin-antidote operons in Wolbachia-induced cytoplasmic incompatibility.
Bajpai, A. and Sinha, P. (2019). Hh signaling from de novo organizers drive lgl neoplasia in Drosophila epithelium. Dev Biol. PubMed ID: 31557471
Summary:
The Hedgehog (Hh) morphogen regulates growth and patterning. Since Hh signaling is also implicated in carcinogenesis, it is conceivable that de novo Hh-secreting organizers, if formed in association with oncogenic hit could be tumor-cooperative. This hypothesis was validated using the Drosophila model of cooperative epithelial carcinogenesis. Somatic clones were generated with simultaneous loss of tumor suppressor, Lgl, and gain of the posterior compartment selector, Engrailed (En), known to induce synthesis of Hh. lgl UAS-en clones in the anterior wing compartment were shown to trigger the Hh signaling cascade via cross-talk with their Ci-expressing wild type cell neighbors. Hh-Dpp signaling from clone boundaries of such ectopically formed de novo organizers in turn drive lgl carcinogenesis. By contrast, Ci-expressing lgl clones transform by autocrine and/or juxtracine activation of Hh signaling in only the posterior compartment. It was further shown that sequestration of the Hh ligand or loss of Dpp receptor, Tkv, in these Hh-sending or -receiving lgl clones arrested their carcinogenesis. These results therefore reveal a hitherto unrecognized mechanism of tumor cooperation by developmental organizers, which are induced fortuitously by oncogenic hits.

Wednesday October 30th - Chromatin

Dorafshan, E., Kahn, T. G., Glotov, A., Savitsky, M. and Schwartz, Y. B. (2019). Genetic dissection reveals the role of Ash1 domains in counteracting Polycomb repression. G3 (Bethesda). PubMed ID: 31540973
Summary:
Antagonistic functions of Polycomb and Trithorax proteins are essential for proper development of all metazoans. While the Polycomb proteins maintain the repressed state of many key developmental genes, the Trithorax proteins ensure that these genes stay active in cells where they have to be expressed. Ash1 is the Trithorax protein that was proposed to counteract Polycomb repression by methylating lysine 36 of histone H3. However, it was recently shown that genetic replacement of Drosophila histone H3 with the variant that carried Arginine instead of Lysine at position 36 did not impair the ability of Ash1 to counteract Polycomb repression. This argues that Ash1 counteracts Polycomb repression by methylating yet unknown substrate(s) and that it is time to look beyond Ash1 methyltransferase SET domain, at other evolutionary conserved parts of the protein that received little attention. This study used Drosophila genetics to demonstrate that Ash1 requires each of the BAH, PHD and SET domains to counteract Polycomb repression, while AT hooks are dispensable. These findings argue that, in vivo, Ash1 acts as a multimer. Thereby it can combine the input of the SET domain and PHD-BAH cassette residing in different peptides. Finally, using new loss of function alleles, zygotic Ash1 was shown to be required to prevent erroneous repression of homeotic genes of the bithorax complex.
Erceg, J., AlHaj Abed, J., Goloborodko, A., Lajoie, B. R., Fudenberg, G., Abdennur, N., Imakaev, M., McCole, R. B., Nguyen, S. C., Saylor, W., Joyce, E. F., Senaratne, T. N., Hannan, M. A., Nir, G., Dekker, J., Mirny, L. A. and Wu, C. T. (2019). The genome-wide multi-layered architecture of chromosome pairing in early Drosophila embryos. Nat Commun 10(1): 4486. PubMed ID: 31582744
Summary:
Genome organization involves cis and trans chromosomal interactions, both implicated in gene regulation, development, and disease. This study focused on trans interactions in Drosophila, where homologous chromosomes are paired in somatic cells from embryogenesis through adulthood. First long-standing questions were addressed regarding the structure of embryonic homolog pairing and, to this end, a haplotype-resolved Hi-C approach was developed to minimize homolog misassignment and thus robustly distinguish trans-homolog from cis contacts. This computational approach, which is called Ohm, reveals pairing to be surprisingly structured genome-wide, with trans-homolog domains, compartments, and interaction peaks, many coinciding with analogous cis features. a significant genome-wide correlation was found between pairing, transcription during zygotic genome activation, and binding of the pioneer factor Zelda. These findings reveal a complex, highly structured organization underlying homolog pairing, first discovered a century ago in Drosophila. Finally, the versatility of this haplotype-resolved approach was demonstrated by applying it to mammalian embryos.
Bughio, F., Huckell, G. R. and Maggert, K. A. (2019). Monitoring of switches in heterochromatin-induced silencing shows incomplete establishment and developmental instabilities. Proc Natl Acad Sci U S A 116(40): 20043-20053. PubMed ID: 31527269
Summary:
Position effect variegation (PEV) in Drosophila results from new juxtapositions of euchromatic and heterochromatic chromosomal regions, and manifests as striking bimodal patterns of gene expression. The semirandom patterns of PEV, reflecting clonal relationships between cells, have been interpreted as gene-expression states that are set in development and thereafter maintained without change through subsequent cell divisions. Many properties of PEV are not predicted from currently accepted biochemical and theoretical models. This work investigated the time at which expressivity of silencing is set and finds that it is determined before heterochromatin exists. A mathematical simulation and a corroborating experimental approach are employed to monitor switching (i.e., gains and losses of silencing) through development. In contrast to current views, this study finds that gene silencing is incompletely set early in embryogenesis, but nevertheless is repeatedly lost and gained in individual cells throughout development. The data support an alternative to locus-specific 'epigenetic' silencing at variegating gene promoters that more fully accounts for the final patterns of PEV.
Bu, B., Chen, L., Zheng, L., He, W. and Zhang, L. (2019). Nipped-A regulates the Drosophila circadian clock via histone deubiquitination. EMBO J: e101259. PubMed ID: 31538360
Summary:
Psychiatric diseases are often accompanied by circadian disruptions, but the molecular underpinnings remain largely unclear. To address this, genes that have been previously reported to be associated with psychiatric diseases were screened, and TRRAP, a gene associated with schizophrenia, was found involved in circadian rhythm regulation. Knocking down Nipped-A, the Drosophila homolog of human TRRAP, leads to lengthened period of locomotor rhythms in flies. Molecular analysis demonstrates that NIPPED-A sets the pace of the clock by increasing the mRNA and protein levels of core clock genes timeless (tim) and Par domain protein 1epsilon (Pdp1epsilon). Furthermore, NIPPED-A promotes the transcription of tim and Pdp1epsilon possibly by facilitating deubiquitination of histone H2B via the deubiquitination module of the transcription co-activator Spt-Ada-Gcn5 acetyltransferase complex. Taken together, these findings reveal a novel role for NIPPED-A in epigenetic regulation of the clock.
Benner, L., Castro, E. A., Whitworth, C., Venken, K. J. T., Yang, H., Fang, J., Oliver, B., Cook, K. R. and Lerit, D. A. (2019). Drosophila heterochromatin stabilization requires the zinc-finger protein Small ovary. Genetics. PubMed ID: 31558581
Summary:
Heterochromatin-mediated repression is essential for controlling the expression of transposons and for coordinated cell type-specific gene regulation. The small ovary (sov) locus was identified in a screen for female-sterile mutations in Drosophila melanogaster, and mutants show dramatic ovarian morphogenesis defects. The null sov phenotype is lethal and maps to the uncharacterized gene CG14438, which encodes a nuclear zinc-finger protein that colocalizes with the essential Heterochromatin Protein 1 (HP1a). Sov functions to repress inappropriate gene expression in the ovary, silence transposons, and suppress position-effect variegation in the eye, suggesting a central role in heterochromatin stabilization.
Chereji, R. V., Bryson, T. D. and Henikoff, S. (2019). Quantitative MNase-seq accurately maps nucleosome occupancy levels. Genome Biol 20(1): 198. PubMed ID: 31519205
Summary:
Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono-nucleosomes, whereas cleavages within nucleosomes destroy them. This study introduces a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms the theory and provides accurate occupancy levels over an MNase digestion time course. As with human cells, no overall differences were observed in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.

Tuesday, October 29th - Transcriptional Regulation

Mouawad, R., Prasad, J., Thorley, D., Himadewi, P., Kadiyala, D., Wilson, N., Kapranov, P. and Arnosti, D. N. (2019). Diversification of retinoblastoma protein function associated with cis and trans adaptations. Mol Biol Evol. PubMed ID: 31418797
Summary:
Retinoblastoma proteins are eukaryotic transcriptional co-repressors that play central roles in cell cycle control, among other functions. Although most metazoan genomes encode a single retinoblastoma protein, gene duplications have occurred at least twice: in the vertebrate lineage, leading to Rb, p107, and p130, and in Drosophila, an ancestral Rbf1 gene and a derived Rbf2 gene. Structurally, Rbf1 resembles p107 and p130, and mutation of the gene is lethal. Rbf2 is more divergent and mutation does not lead to lethality. However, the retention of Rbf2 over 60 million years in Drosophila points to essential functions, which prior cell-based assays have been unable to elucidate. Using genomic approaches, this study provides new insights on the function of Rbf2. Strikingly, Rbf2 was shown to regulate a set of cell growth related genes and can antagonize Rbf1 on specific genes. These unique properties have important implications for the fly; Rbf2 mutants show reduced egg laying, and lifespan is reduced in females and males. Structural alterations in conserved regions of Rbf2 gene suggest that it was sub- or neofunctionalized to develop specific regulatory specificity and activity. This study defines cis regulatory features of Rbf2 target genes that allow preferential repression by this protein, indicating that it is not a weaker version of Rbf1 as previously thought. The specialization of retinoblastoma function in Drosophila may reflect a parallel evolution found in vertebrates, and raises the possibility that cell growth control is equally important to cell cycle function for this conserved family of transcriptional corepressors.
Delker, R. K., Ranade, V., Loker, R., Voutev, R. and Mann, R. S. (2019). Low affinity binding sites in an activating CRM mediate negative autoregulation of the Drosophila Hox gene Ultrabithorax. PLoS Genet 15(10): e1008444. PubMed ID: 31589607
Summary:

Specification of cell identity and the proper functioning of a mature cell depend on precise regulation of gene expression. Both binary ON/OFF regulation of transcription, as well as more fine-tuned control of transcription levels in the ON state, are required to define cell types. The Drosophila melanogaster Hox gene, Ultrabithorax (Ubx), exhibits both of these modes of control during development. While ON/OFF regulation is needed to specify the fate of the developing wing (Ubx OFF) and haltere (Ubx ON), the levels of Ubx within the haltere differ between compartments along the proximal-distal axis. This study identified and molecularly dissected the novel contribution of a previously identified Ubx cis-regulatory module (CRM), anterobithorax (abx), to a negative auto-regulatory loop that decreases Ubx expression in the proximal compartment of the haltere as compared to the distal compartment. Ubx, in complex with the known Hox cofactors, Homothorax (Hth) and Extradenticle (Exd), acts through low-affinity Ubx-Exd binding sites to reduce the levels of Ubx transcription in the proximal compartment. Importantly, Ubx-Exd-binding site mutations sufficient to result in de-repression of abx activity in a transgenic context are not sufficient to de-repress Ubx expression when mutated at the endogenous locus, suggesting the presence of multiple mechanisms through which Ubx-mediated repression occurs. These results underscore the complementary nature of CRM analysis through transgenic reporter assays and genome modification of the endogenous locus; but, they also highlight the increasing need to understand gene regulation within the native context to capture the potential input of multiple genomic elements on gene control.

Falo-Sanjuan, J., Lammers, N. C., Garcia, H. G. and Bray, S. J. (2019). Enhancer priming enables fast and sustained transcriptional responses to Notch signaling. Dev Cell 50(4): 411-425. PubMed ID: 31378591
Summary:
Information from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2-MCP system to visualize nascent transcripts in single cells in Drosophila embryos, it was revealed how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers, two key principles were uncovered. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.
Garcia-Marques, J., Yang, C. P., Espinosa-Medina, I., Mok, K., Koyama, M. and Lee, T. (2019). Unlimited genetic switches for cell-type-specific manipulation. Neuron. PubMed ID: 31395429
Summary:

Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed profiling of cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. This paper presents CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific guide RNA (gRNA). Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

Ramaekers, A., Claeys, A., Kapun, M., Mouchel-Vielh, E., Potier, D., Weinberger, S., Grillenzoni, N., Dardalhon-Cumenal, D., Yan, J., Wolf, R., Flatt, T., Buchner, E. and Hassan, B. A. (2019). Altering the temporal regulation of one transcription factor drives evolutionary trade-Offs between head sensory organs. Dev Cell. PubMed ID: 31447264
Summary:
Size trade-offs of visual versus olfactory organs is a pervasive feature of animal evolution. This could result from genetic or functional constraints. This study demonstrates that head sensory organ size trade-offs in Drosophila are genetically encoded and arise through differential subdivision of the head primordium into visual versus non-visual fields. Changes were discovered in the temporal regulation of the highly conserved eyeless/Pax6 gene expression during development is a conserved mechanism for sensory trade-offs within and between Drosophila species. A natural single nucleotide polymorphism was identified in the cis-regulatory region of eyeless in a binding site of its repressor Cut that is sufficient to alter its temporal regulation and eye size. Because eyeless/Pax6 is a conserved regulator of head sensory placode subdivision, it is proposed that its temporal regulation is key to define the relative size of head sensory organs.
Sanchez-Higueras, C., Rastogi, C., Voutev, R., Bussemaker, H. J., Mann, R. S. and Hombria, J. C. (2019). In vivo Hox binding specificity revealed by systematic changes to a single cis regulatory module. Nat Commun 10(1): 3597. PubMed ID: 31399572
Summary:
Hox proteins belong to a family of transcription factors with similar DNA binding specificities that control animal differentiation along the antero-posterior body axis. Hox proteins are expressed in partially overlapping regions where each one is responsible for the formation of particular organs and structures through the regulation of specific direct downstream targets. Thus, explaining how each Hox protein can selectively control its direct targets from those of another Hox protein is fundamental to understand animal development. This study analyzed a cis regulatory module directly regulated by seven different Drosophila Hox proteins and uncovered how different Hox class proteins differentially control its expression. Regulation by one or another Hox protein depends on the combination of three modes: Hox-cofactor dependent DNA-binding specificity; Hox-monomer binding sites; and interaction with positive and negative Hox-collaborator proteins. Additionally, this study found that similar regulation can be achieved by Amphioxus orthologs, suggesting these three mechanisms are conserved from insects to chordates.

Monday, October 28th - Cell Cycle

Rotelli, M. D., Policastro, R. A., Bolling, A. M., Killion, A. W., Weinberg, A. J., Dixon, M. J., Zentner, G. E., Walczak, C. E., Lilly, M. A. and Calvi, B. R. (2019). A Cyclin A-Myb-MuvB-Aurora B network regulates the choice between mitotic cycles and polyploid endoreplication cycles. PLoS Genet 15(7): e1008253. PubMed ID: 31291240
Summary:
Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. This study used integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs). The evidence suggests that Cyclin A / CDK directly activates the Myb-MuvB (MMB) complex to induce transcription of a battery of genes required for mitosis, and that repression of CDK activity dampens this MMB mitotic transcriptome to promote endoreplication in both iECs and devECs. iECs and devECs differed, however, in that devECs had reduced expression of E2f1-dependent genes that function in S phase, whereas repression of the MMB transcriptome in iECs was sufficient to induce endoreplication without a reduction in S phase gene expression. Among the MMB regulated genes, knockdown of AurB protein and other subunits of the chromosomal passenger complex (CPC) induced endoreplication, as did knockdown of CPC-regulated cytokinetic, but not kinetochore, proteins. Together, these results indicate that the status of a CycA-Myb-MuvB-AurB network determines the decision to commit to mitosis or switch to endoreplication in both iECs and devECs, and suggest that regulation of different steps of this network may explain the known diversity of polyploid cycle types in development and disease.
Harding, K. and White, K. (2019). Decoupling developmental apoptosis and neuroblast proliferation in Drosophila. Dev Biol. PubMed ID: 31390535
Summary:
Cell proliferation and cell death are opposing but fundamental aspects of development that must be tightly controlled to ensure proper tissue organization and organismal health. Developmental apoptosis of abdominal neuroblasts in the Drosophila ventral nerve cord is controlled by multiple upstream spatial and temporal signals, which have also been implicated in control of cell proliferation. It has therefore remained unclear whether developmental apoptosis is linked to active cell proliferation. Previous investigations into this topic have focused on the effect of cell cycle arrests on exogenous induction of apoptosis, and thus have not addressed whether potential effects of the cell cycle lie with the sensing of damage signals or the execution of apoptosis itself. This report shows that developmental apoptosis is not inhibited by cell cycle arrest, and that endogenous cell death occurs independently of cell cycle phase. Ectopic neuroblasts rescued from cell death retain the competency to respond to quiescence cues at the end of embryogenesis. In addition, multiple quiescence types were observed in neuroblasts, and cell death mutant embryos display a specific loss of presumptive G2 quiescent abdominal neuroblasts at the end of embryogenesis. This study demonstrates that upstream control of neuroblast proliferation and apoptosis represent independent mechanisms of regulating stem cell fate, and that execution of apoptosis occurs in a cell cycle-independent manner. These findings also indicate that a subset of G2Q-fated abdominal neuroblasts are eliminated from the embryo through a non-apoptotic mechanism.
Afonso, O., Castellani, C. M., Cheeseman, L. P., Ferreira, J. G., Orr, B., Ferreira, L. T., Chambers, J. J., Morais-de-Sa, E., Maresca, T. J. and Maiato, H. (2019). Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk. Elife 8. PubMed ID: 31424385
Summary:
According to the prevailing 'clock' model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the 'ruler' model. This study found that Cdk1 remains active during anaphase due to ongoing APC/C(Cdc20)- and APC/C(Cdh1)-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular 'rulers' and 'clocks' licenses mitotic exit only after proper chromosome separation.
Billmyre, K. K., Cahoon, C. K., Heenan, G. M., Wesley, E. R., Yu, Z., Unruh, J. R., Takeo, S. and Hawley, R. S. (2019). X chromosome and autosomal recombination are differentially sensitive to disruptions in SC maintenance. Proc Natl Acad Sci U S A. PubMed ID: 31570610
Summary:
The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double-strand breaks (DSBs) into crossovers or gene conversions. The removal of any central-region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, CRISPR/Cas9 was used to construct 3 in-frame deletions within the predicted coiled-coil region of the C(3)G protein. Since these 3 deletion mutations disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, they allowed definition the stages of pachytene when the SC is necessary for homolog pairing and recombination during pachytene. These studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently from the autosomes.
Costa, M. F. A. and Ohkura, H. (2019). The molecular architecture of the meiotic spindle is remodeled during metaphase arrest in oocytes. J Cell Biol. PubMed ID: 31278080
Summary:
Before fertilization, oocytes of most species undergo a long, natural arrest in metaphase. Before this, prometaphase I is also prolonged, due to late stable kinetochore-microtubule attachment. How oocytes stably maintain the dynamic spindle for hours during these periods is poorly understood. This study reports that the bipolar spindle changes its molecular architecture during the long prometaphase/metaphase I in Drosophila melanogaster oocytes. By generating transgenic flies expressing GFP-tagged spindle proteins, it was found that 14 of 25 spindle proteins change their distribution in the bipolar spindle. Among them, microtubule cross-linking kinesins, MKlp1/Pavarotti and kinesin-5/Klp61F, accumulate to the spindle equator in late metaphase. The late equator accumulation of MKlp1/Pavarotti is regulated by a mechanism distinct from that in mitosis. While MKlp1/Pavarotti contributes to the control of spindle length, kinesin-5/Klp61F is crucial for maintaining a bipolar spindle during metaphase I arrest. This study provides novel insight into how oocytes maintain a bipolar spindle during metaphase arrest.
Grendler, J., Lowgren, S., Mills, M. and Losick, V. P. (2019). Wound-induced polyploidization is driven by Myc and supports tissue repair in the presence of DNA damage. Development 146(15). PubMed ID: 31315896
Summary:
Tissue repair usually requires either polyploid cell growth or cell division, but the molecular mechanism promoting polyploidy and limiting cell division remains poorly understood. This study finds that injury to the adult Drosophila epithelium causes cells to enter the endocycle through the activation of Yorkie-dependent genes (Myc and E2f1). Myc is even sufficient to induce the endocycle in the uninjured post-mitotic epithelium. As result, epithelial cells enter S phase but mitosis is blocked by inhibition of mitotic gene expression. The mitotic cell cycle program can be activated by simultaneously expressing the Cdc25-like phosphatase String (stg), while genetically depleting APC/C E3 ligase fizzy-related (fzr). However, forcing cells to undergo mitosis is detrimental to wound repair as the adult fly epithelium accumulates DNA damage, and mitotic errors ensue when cells are forced to proliferate. In conclusion, this study found that wound-induced polyploidization enables tissue repair when cell division is not a viable option.

Friday, October 25th

Spracklen, A. J., Lamb, M. C., Groen, C. M. and Tootle, T. L. (2019). Pharmaco-genetic screen to uncover actin regulators targeted by prostaglandins during Drosophila oogenesis. G3 (Bethesda). PubMed ID: 31506320
Summary:
Prostaglandins (PGs) are lipid signaling molecules with numerous physiologic functions, including pain/inflammation, fertility, and cancer. PGs are produced downstream of cyclooxygenase (COX) enzymes, the targets of non-steroidal anti-inflammatory drugs (NSAIDs). In numerous systems, PGs regulate actin cytoskeletal remodeling, however, their mechanisms of action remain largely unknown. To address this deficiency, a pharmaco-genetic interaction screen was undertaken during late-stage Drosophila oogenesis. Drosophila oogenesis is as an established model for studying both actin dynamics and PGs. Indeed, during Stage 10B, cage-like arrays of actin bundles surround each nurse cell nucleus, and during Stage 11, the cortical actin contracts, squeezing the cytoplasmic contents into the oocyte. Both of these cytoskeletal properties are required for follicle development and fertility, and are regulated by PGs. This study describes a pharmaco-genetic interaction screen that takes advantage of the fact that Stage 10B follicles will mature in culture and COX inhibitors, such as aspirin, block this in vitro follicle maturation. In the screen, aspirin was used at a concentration that blocks 50% of the wild-type follicles from maturing in culture. By combining this aspirin treatment with heterozygosity for mutations in actin regulators, enhancers and suppressors of COX inhibition were quantitatively identified. This study presents the screen results and initial follow-up studies on three strong enhancers - Enabled, Capping protein, and non-muscle Myosin II Regulatory Light Chain. Overall, these studies provide new insight into how PGs regulate both actin bundle formation and cellular contraction, properties that are not only essential for development, but are misregulated in disease.
Smith, P., Buhl, E., Tsaneva-Atanasova, K. and Hodge, J. J. L. (2019). Shaw and Shal voltage-gated potassium channels mediate circadian changes in Drosophila clock neuron excitability. J Physiol. PubMed ID: 31612994
Summary:
Like in mammals, Drosophila circadian clock neurons display rhythms of activity with higher action potential firing rates and more positive resting membrane potentials during the day. This rhythmic excitability has been widely observed but, critically, its regulation remains unresolved. This study characterized and modeled the changes underlying these electrical activity rhythms in the lateral ventral clock neurons (LNvs). Currents mediated by the voltage-gated potassium channels Shaw (Kv3) and Shal (Kv4) oscillate in a circadian manner. Disruption of these channels, by expression of dominant negative (DN) subunits, leads to changes in circadian locomotor activity and shortens lifespan. LNv whole-cell recordings then show that changes in Shaw and Shal currents drive changes in action potential firing rate and that these rhythms are abolished when the circadian molecular clock is stopped. A whole-cell biophysical model using Hodgkin-Huxley equations can recapitulate these changes in electrical activity. Based on this model and by using dynamic clamp to manipulate clock neurons directly, the pharmacological block of Shaw and Shal can be rescued, restoring the firing rhythm, and thus demonstrating the critical importance of Shaw and Shal. Together, these findings point to a key role for Shaw and Shal in controlling circadian firing of clock neurons and show that changes in clock neuron currents can account for this. Moreover, with dynamic clamp the LNvs between morning-like and evening-like states of electrical activity can be switched. It is concluded that changes in Shaw and Shal underlie the daily oscillation in LNv firing rate.
Stanley, C. E., Mauss, A. S., Borst, A. and Cooper, R. L. (2019). The effects of chloride flux on Drosophila heart rate. Methods Protoc 2(3). PubMed ID: 31443492
Summary:
Approaches are sought after to regulate ionotropic and chronotropic properties of the mammalian heart. Electrodes are commonly used for rapidly exciting cardiac tissue and resetting abnormal pacing. With the advent of optogenetics and the use of tissue-specific expression of light-activated channels, cardiac cells cannot only be excited but also inhibited with ion-selective conductance. As a proof of concept for the ability to slow down cardiac pacing, anion-conducting channelrhodopsins (GtACR1/2) and the anion pump halorhodopsin (eNpHR) were expressed in hearts of larval Drosophila and activated by light. Unlike body wall muscles in most animals, the equilibrium potential for Cl(-) is more positive as compared to the resting membrane potential in larval Drosophila. As a consequence, upon activating the two forms of GtACR1 and 2 with low light intensity the heart rate increased, likely due to depolarization and opening of voltage-gated Ca(2+) channels. However, with very intense light activation the heart rate ceases, which may be due to Cl(-) shunting to the reversal potential for chloride. Activating eNpHR hyperpolarizes body wall and cardiac muscle in larval Drosophila and rapidly decreases heart rate. The decrease in heart rate is related to light intensity. Intense light activation of eNpHR stops the heart from beating, whereas lower intensities slowed the rate. Even with upregulation of the heart rate with serotonin, the pacing of the heart was slowed with light. Thus, regulation of the heart rate in Drosophila can be accomplished by activating anion-conducting channelrhodopsins using light. These approaches are demonstrated in a genetically amenable insect model.
Sorensen, J. G., Giribets, M. P., Tarrio, R., Rodriguez-Trelles, F., Schou, M. F. and Loeschcke, V. (2019). Expression of thermal tolerance genes in two Drosophila species with different acclimation capacities. J Therm Biol 84: 200-207. PubMed ID: 31466754
Summary:
Heat tolerance increases at higher acclimation temperatures in D. melanogaster, but not in D. subobscura. The two species represent separate lineages of the subgenus Sophophora of Drosophila with contrasting tropical African and temperate Palearctic evolutionary histories. D. melanogaster has five copies of the inducible hsp70 gene distributed in two clusters, named A (with two copies) and B (three copies), while D. subobscura has only two copies arranged similarly to cluster A of D. melanogaster. The hsp70s of the two species also differ in their cis-regulatory regions, with D. melanogaster exhibiting features of a faster and more productive promoter. It was predicted that the interspecific variation in acclimation capacity of heat tolerance is explained by evolved variation in expression of the major group of heat shock proteins. To test this prediction, basal levels of gene expression were compared at different developmental temperatures within each of the two species. Furthermore, the heat hardening dynamics were explored by measuring the induction of gene expression during a ramping assay. The prediction of a stronger heat shock protein response in D. melanogaster as compared to D. subobscura was confirmed for both long-term acclimation and short-term hardening. For D. melanogaster the upregulation with temperature ramping ranged from less than two fold (hsp26) to 2500 fold (hsp70A) increase. In all cases induction in D. melanogaster exceeded that of D. subobscura homologs. These differences correlate with structural differences in the regulatory regions of hsp70, and might explain differences in acclimation capacity among species. Finally, in D. melanogaster an indication was found of an inverse relationship between basal and induced levels of hsp70A and hsp83 expression, suggesting a divergent role for thermal adaptation of these genes at benign and stressful temperatures, respectively.
Wilinski, D., Winzeler, J., Duren, W., Persons, J. L., Holme, K. J., Mosquera, J., Khabiri, M., Kinchen, J. M., Freddolino, P. L., Karnovsky, A. and Dus, M. (2019). Rapid metabolic shifts occur during the transition between hunger and satiety in Drosophila melanogaster. Nat Commun 10(1): 4052. PubMed ID: 31492856
Summary:
Metabolites are active controllers of cellular physiology, but their role in complex behaviors is less clear. This study reports metabolic changes that occur during the transition between hunger and satiety in Drosophila melanogaster. To analyze these data in the context of fruit fly metabolic networks, this study developed Flyscape, an open-access tool. In response to eating, metabolic profiles change in quick, but distinct ways in the heads and bodies. Consumption of a high sugar diet dulls the metabolic and behavioral differences between the fasted and fed state and reshapes the way nutrients are utilized upon eating. Specifically, high dietary sugar was found to increase TCA cycle activity, alter neurochemicals, and deplete 1-carbon metabolism and brain health metabolites N-acetyl-aspartate and kynurenine. Together, this work identifies the metabolic transitions that occur during hunger and satiation, and provides a platform to study the role of metabolites and diet in complex behavior.
Weismann, C. G., Blice-Baum, A., Tong, T., Li, J., Huang, B. K., Jonas, S. M., Cammarato, A. and Choma, M. A. (2019). Multi-modal and multiscale imaging approaches reveal novel cardiovascular pathophysiology in Drosophila melanogaster. Biol Open 8(8). PubMed ID: 31455664
Summary:
This study reports a comprehensive assessmentment of cardiovascular physiology in Drosophila. High-speed angiography, optical coherence tomography (OCT) and confocal microscopy were employed to reveal functional and structural abnormalities in the hdp(2) mutant, pre-pupal heart tube and aorta relative to controls. hdp(2) harbors a mutation in wupA, which encodes an ortholog of human troponin I (TNNI3). TNNI3 variants frequently engender cardiomyopathy. The hdp(2) aortic and cardiac muscle walls are disrupted and that shorter sarcomeres are associated with smaller, stiffer aortas, which consequently result in increased flow and pulse wave velocities. The mutant hearts also displayed diastolic and latent systolic dysfunction. It is concluded that hdp(2) pre-pupal hearts are exposed to increased afterload due to aortic hypoplasia. This may in turn contribute to diastolic and subtle systolic dysfunction via vascular-heart tube interaction, which describes the effect of the arterial loading system on cardiac function. This study demonstrates that complex and dynamic micro- and mesoscopic phenotypes can be mechanistically explained in a gene sequence- and molecular-specific manner.

Thursday, October 24th - Disease Models

Wen, D. T., Zheng, L., Li, J. X., Lu, K. and Hou, W. Q. (2019). The activation of cardiac dSir2-related pathways mediates physical exercise resistance to heart aging in old Drosophila. Aging (Albany NY) 11. PubMed ID: 31503544
Summary:
Cardiac aging is notably characterized by increased diastolic dysfunction, lipid accumulation, oxidative stress, and contractility debility. The Sir2/Sirt1 gene overexpression delays cell aging and reduces obesity and oxidative stress. Exercise improves heart function and delays heart aging. However, it remains unclear whether exercise delaying heart aging is related to cardiac Sir2/Sirt1-related pathways. In this study, cardiac dSir2 overexpression or knockdown was regulated using the UAS/hand-Gal4 system in Drosophila. Flies underwent exercise interventions from 4 weeks to 5 weeks old. Results showed that either cardiac dSir2 overexpression or exercise remarkably increased the cardiac period, systolic interval, diastolic interval, fractional shortening, SOD activity, dSIR2 protein, Foxo, dSir2, Nmnat, and bmm expression levels in the aging flies; they also notably reduced the cardiac triacylglycerol level, malonaldehyde level, and the diastolic dysfunction index. Either cardiac dSir2 knockdown or aging had almost opposite effects on the heart as those of cardiac dSir2 overexpression. Therefore, this study claims that cardiac dSir2 overexpression or knockdown delayed or promoted heart aging by reducing or increasing age-related oxidative stress, lipid accumulation, diastolic dysfunction, and contractility debility. The activation of cardiac dSir2/Foxo/SOD and dSir2/Foxo/Bmm pathways may be two important molecular mechanisms through which exercise works against heart aging in Drosophila.
Brischigliaro, M., Corra, S., Tregnago, C., Fernandez-Vizarra, E., Zeviani, M., Costa, R. and De Pitta, C. (2019). Knockdown of APOPT1/COA8 causes Cytochrome c oxidase deficiency, neuromuscular impairment, and reduced resistance to oxidative stress in Drosophila melanogaster. Front Physiol 10: 1143. PubMed ID: 31555154
Summary:
Cytochrome c oxidase (COX) deficiency is the biochemical hallmark of several mitochondrial disorders, including subjects affected by mutations in apoptogenic-1 (APOPT1), recently renamed as COA8 (HGNC:20492). Loss-of-function mutations are responsible for a specific infantile or childhood-onset mitochondrial leukoencephalopathy with a chronic clinical course. Patients deficient in COA8 show specific COX deficiency with distinctive neuroimaging features, i.e., cavitating leukodystrophy. In human cells, COA8 is rapidly degraded by the ubiquitin-proteasome system, but oxidative stress stabilizes the protein, which is then involved in COX assembly, possibly by protecting the complex from oxidative damage. However, its precise function remains unknown. The CG14806 gene (dCOA8) is the Drosophila melanogaster ortholog of human COA8 encoding a highly conserved COA8 protein. This study reportd that dCOA8 knockdown (KD) flies show locomotor defects, and other signs of neurological impairment, reduced COX enzymatic activity, and reduced lifespan under oxidative stress conditions. These data indicate that KD of dCOA8 in Drosophila phenocopies several features of the human disease, thus being a suitable model to characterize the molecular function/s of this protein in vivo and the pathogenic mechanisms associated with its defects.
Wakisaka, K. T., Muraoka, Y., Shimizu, J., Yamaguchi, M., Ueoka, I., Mizuta, I., Yoshida, H. and Yamaguchi, M. (2019). Drosophila Alpha-ketoglutarate-dependent dioxygenase AlkB is involved in repair from neuronal disorders induced by ultraviolet damage. Neuroreport. PubMed ID: 31503204
Summary:
AlkB family proteins are enzymes that repair alkylated DNA and RNA by oxidative demethylation. Nine homologs have been identified and characterized in mammals. ALKBH1 is conserved among metazoans including Drosophila. Although the ALKBH1 mouse homolog, Alkbh1 functions in neurogenesis, it currently remains unclear whether ALKBH1 plays a role in neuronal disorders induced by ultraviolet-induced DNA damage. This study has demonstrated that the Drosophila ALKBH1 homolog, AlkB contributed to recovery from neuronal disorders induced by ultraviolet damage. The knockdown of AlkB resulted in not only learning defects but also altered crawling behavior in Drosophila larvae after ultraviolet irradiation. A molecular analysis revealed that AlkB contributed to the repair of ultraviolet-induced DNA damage in the central nervous system of larvae. Therefore, it is proposed that ALKBH1 plays a role in the repair of ultraviolet-induced DNA damage in central nervous system. Ultraviolet-induced DNA damage is involved in the pathogenesis of xeroderma pigmentosum, and has recently been implicated in Parkinson's disease. The present results will contribute to understanding of neuronal diseases induced by ultraviolet-induced DNA damage.
Russo, A. and DiAntonio, A. (2019). Wnd/DLK is a critical target of FMRP responsible for neurodevelopmental and behavior defects in the Drosophila model of Fragile X syndrome. Cell Rep 28(10): 2581-2593. PubMed ID: 31484070
Summary:
Fragile X syndrome (FXS) is the leading heritable cause of intellectual disability and commonly co-occurs with autism spectrum disorder. Silencing of the Fmr1 gene leads to the absence of the protein product, fragile X mental retardation protein (FMRP), which represses translation of many target mRNAs. Excess translation of these targets is one cause of neuronal dysfunction in FXS. Utilizing the Drosophila model of FXS, this study identified the mitogen-activated protein kinase kinase kinase (MAP3K) Wallenda/dual leucine zipper kinase (DLK) as a critical target of FMRP. dFMRP binds Wallenda mRNA and is required to limit Wallenda protein levels. In dFmr1 mutants, Wallenda signaling drives defects in synaptic development, neuronal morphology, and behavior. Pharmacological inhibition of Wallenda in larvae suppresses dFmr1 neurodevelopmental phenotypes, while adult administration prevents dFmr1 behavioral defects. It is proposed that in dFmr1 mutants chronic Wallenda/DLK signaling disrupts nervous system development and function and that inhibition of this kinase cascade might be a candidate therapeutic intervention for the treatment of FXS.
Tower, J., Agrawal, S., Alagappan, M. P., Bell, H. S., Demeter, M., Havanoor, N., Hegde, V. S., Jia, Y., Kothawade, S., Lin, X., Nadig, C., Rajashekharappa, N. S., Rao, D., Rao, S. S., Sancheti, P., Saria, A., Shantharamu, N. H., Sharma, V., Tadepalli, K. and Varma, A. (2019). Behavioral and molecular markers of death in Drosophila melanogaster. Exp Gerontol 126: 110707. PubMed ID: 31445108
Summary:
Fly movement was tracked through 3-dimensional (3D) space as the fly died, using either reflected visible light, reflected infrared (IR) light, or fly GFP fluorescence. Behaviors measured included centrophobism, negative geotaxis, velocity, and total activity. In addition, frequency of directional heading changes (FDHC) was calculated as a measure of erratic movement. Nine middle-aged flies were tracked as they died during normal aging, and fifteen young flies were tracked as they died from dehydration/starvation stress. Episodes of increased FDHC were observed 0-8h prior to death for the majority of the flies. FDHC was also increased with age in flies with neuronal expression of a human Abeta42 protein fragment associated with Alzheimer's disease. Finally, green autofluorescence appeared in the eye and body immediately prior to and coincident with death, and fluorescence of GFP targeted to the retina increased immediately prior to and coincident with death. The results suggest the potential utility of FDHC, green autofluorescence, and retinal GFP as markers of neuronal malfunction and imminent death.
Scepanovic, G. and Stewart, B. A. (2019). Analysis of Drosophila nervous system development following an early, brief exposure to ethanol. Dev Neurobiol. PubMed ID: 31472090
Summary:
The effects of ethanol on neural function and development have been studied extensively, motivated in part by the addictive properties of alcohol and the neurodevelopmental deficits that arise in children with Fetal Alcohol Spectrum Disorder (FASD). Absent from this research area is a genetically tractable system to study the effects of early ethanol exposure on later neurodevelopmental and behavioural phenotypes. Embryos of the fruit fly, Drosophila melanogaster have been used as a model system to investigate the neuronal defects that arise after an early exposure to ethanol. Several disruptions of neural development and morphology were found following a brief ethanol exposure during embryogenesis along with subsequent changes in larval behaviour. Altogether, this study establishes a new system to examine the effects of alcohol exposure in embryos and the potential to conduct large scale genetics screens to uncover novel factors that sensitize or protect neurons to the effects of alcohol.

Wednesday, October 23rd - Evolution

Kauranen, H., Kinnunen, J., Hiillos, A. L., Lankinen, P., Hopkins, D., Wiberg, R. A. W., Ritchie, M. G. and Hoikkala, A. (2019). Selection for reproduction under short photoperiods changes diapause-associated traits and induces widespread genomic divergence. J Exp Biol. PubMed ID: 31511345
Summary:
The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are interrelated is not well understood. This study performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana, to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and / or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence between the selection and control line replicates in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled ones detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, this study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity.
Leach, H., Van Timmeren, S., Wetzel, W. and Isaacs, R. (2019). Predicting within- and between-year variation in activity of the invasive spotted wing Drosophila (Diptera: Drosophilidae) in a temperate region. Environ Entomol. PubMed ID: 31502634
Summary:
Invasive insect pests can be challenging to manage because their recent arrival provides limited information on which to build predictive population models. The magnitude and timing of activity by the invasive vinegar fly, Drosophila suzukii, in crop fields has been unpredictable due to its recent arrival in many new regions of the world and changes in methods for its detection. Using 7 yr of consistent trapping of adults at four blueberry farms in Michigan, United States, this study modeled the temporal and environmental factors influencing D. suzukii activity. This pest established high levels within 2 yr of being detected, with peak fly activity continuing to increase. Fly activity timing and abundance were predicted by the annual number of days below 0 degrees C, the number of winter and spring days above 10 degrees C, and by the fly activity in the preceding year, providing support for overwintering in this region. Larval infestation was monitored for 4 yr at these same sites, and a moderate positive correlation was found between larvae in fruit and adults in traps. Finally, a generalized additive model was developed to predict D. suzukii fly capture throughout the season based on relevant environmental factors, and the relative timing and magnitude of activity was examined under varying winter and spring temperature conditions. The results suggest that D. suzukii activity is predictable and that environmental conditions can be used in temperate regions to provide regional risk warnings as a component of strategies to manage this invasive insect pest.
Watanabe, K., Kanaoka, Y., Mizutani, S., Uchiyama, H., Yajima, S., Watada, M., Uemura, T. and Hattori, Y. (2019). Interspecies comparative analyses reveal distinct carbohydrate-responsive systems among Drosophila species. Cell Rep 28(10): 2594-2607. PubMed ID: 31484071
Summary:
During evolution, organisms have acquired variable feeding habits. Some species are nutritional generalists that adapt to various food resources, while others are specialists, feeding on specific resources. However, much remains to be discovered about how generalists hhhadapt to diversified diets. Larvae of the generalists Drosophila melanogaster and D. simulans develop on three diets with different nutrient balances, whereas specialists D. sechellia and D. elegans cannot develop on carbohydrate-rich diets. The generalist D. melanogaster downregulates the expression of diverse metabolic genes systemically by transforming growth factor beta (TGF-beta)/Activin signaling, maintains metabolic homeostasis, and successfully adapts to the diets. In contrast, the specialist D. sechellia expresses those metabolic genes at higher levels and accumulates various metabolites on the carbohydrate-rich diet, culminating in reduced adaptation. Phenotypic similarities and differences strongly suggest that the robust carbohydrate-responsive regulatory systems are evolutionarily retained through genome-environment interactions in the generalists and contribute to their nutritional adaptabilities.
Taylor, S. E., Tuffery, J., Bakopoulos, D., Lequeux, S., Warr, C. G., Johnson, T. K. and Dearden, P. K. (2019). The torso-like gene functions to maintain the structure of the vitelline membrane in Nasonia vitripennis, implying its co-option into Drosophila axis formation. Biol Open. PubMed ID: 31488408
Summary:
Axis specification in Drosophila occurs via the localized activation of the receptor tyrosine kinase Torso. In Hymenoptera however, the same process appears to be achieved via localized mRNA. How these mechanisms evolved and what they evolved from remains largely unexplored. This study shows that torso-like, known for its role in Drosophila terminal patterning, is instead required for the integrity of the vitelline membrane in the hymenopteran wasp Nasonia vitripennis. Other genes known to be involved in Drosophila terminal patterning, such as torso and Ptth, also do not function in Nasonia embryonic development. These findings extended to orthologues of Drosophila vitelline membrane proteins known to play a role in localizing Torso-like in Drosophila; in Nasonia these are instead required for dorso-ventral patterning, gastrulation, and potentially terminal patterning. The data underscores the importance of the vitelline membrane in insect development, and implies phenotypes caused by knockdown of torso-like must be interpreted in light of its function in the vitelline membrane. In addition, the data implies that the signalling components of the Drosophila terminal patterning systems were co-opted from roles in regulating moulting, and co-option into terminal patterning involved the evolution of a novel interaction with the vitelline membrane protein Torso-like.
Kinzner, M. C., Gamisch, A., Hoffmann, A. A., Seifert, B., Haider, M., Arthofer, W., Schlick-Steiner, B. C. and Steiner, F. M. (2019). Major range loss predicted from lack of heat adaptability in an alpine Drosophila species. Sci Total Environ 695: 133753. PubMed ID: 31425981
Summary:
Climate warming is threatening biodiversity worldwide. Climate specialists such as alpine species are especially likely to be vulnerable. Adaptation by rapid evolution is the only long-term option for survival of many species, but the adaptive evolutionary potential of heat resistance has not been assessed in an alpine invertebrate. This study show that the alpine fly Drosophila nigrosparsa cannot readily adapt to heat stress. Heat-exposed flies from a regime with increased ambient temperature and a regime with increased temperature plus artificial selection for heat tolerance were less heat tolerant than the control group. Increased ambient temperature affected negatively both fitness and competitiveness. Ecological niche models predicted the loss of three quarters of the climatically habitable areas of this fly by the end of this century. These findings suggest that, alongside with other climate specialists, species from mountainous regions are highly vulnerable to climate warming and unlikely to adapt through evolutionary genetic changes.
Taverner, A. M., Yang, L., Barile, Z. J., Lin, B., Peng, J., Pinharanda, A. P., Rao, A. S., Roland, B. P., Talsma, A. D., Wei, D., Petschenka, G., Palladino, M. J. and Andolfatto, P. (2019). Adaptive substitutions underlying cardiac glycoside insensitivity in insects exhibit epistasis in vivo. Elife 8. PubMed ID: 31453806
Summary:
Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. This study used genome engineering of Drosophila to investigate constraints on the repeated evolution of unrelated herbivorous insects to toxic cardiac glycosides, which primarily occurs via a small subset of possible functionally-relevant substitutions to Na(+),K(+)-ATPase. Surprisingly, frequently observed adaptive substitutions were found at at two sites, 111 and 122, are lethal when homozygous and adult heterozygotes exhibit dominant neural dysfunction. A phylogenetically correlated substitution, A119S, was found that partially ameliorates the deleterious effects of substitutions at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro, A119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. Tgese results demonstrate the importance of epistasis in constraining adaptive paths. Moreover, by revealing distinct effects of substitutions in vitro and in vivo, the results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms.

Tuesday, October 22nd - Chromatin

Maksimov, D. A. and Koryakov, D. E. (2019). Binding of SU(VAR)3-9 partially depends on SETDB1 in the chromosomes of Drosophila melanogaster. Cells 8(9). PubMed ID: 31491894
Summary:
H3K9 methylation is known to play a critical role in gene silencing. This modification is established and maintained by several enzymes, but relationships between them are not fully understood. This study decipher the interplay between two Drosophila H3K9-specific histone methyltransferases, SU(VAR)3-9 and SETDB1. It was asked whether SETDB1 is required for targeting of SU(VAR)3-9. Using DamID-seq, SU(VAR)3-9 binding profiles were obtained for the chromosomes from larval salivary glands and germline cells from adult females, and profiles were compared between the wild type and SETDB1-mutant backgrounds. These analyses indicate that the vast majority of single copy genes in euchromatin are targeted by SU(VAR)3-9 only in the presence of SETDB1, whereas SU(VAR)3-9 binding at repeated sequences in heterochromatin is largely SETDB1-independent. Interestingly, piRNA clusters 42AB and 38C in salivary gland chromosomes bind SU(VAR)3-9 regardless of SETDB1, whereas binding to the same regions in the germline cells is SETDB1-dependent. In addition, SU(VAR)3-9 profiles were compared in female germline cells at different developmental stages (germarium cells in juvenile ovaries and mature nurse cells). It turned out that SU(VAR)3-9 binding is influenced both by the presence of SETDB1, as well as by the differentiation stage.
Paddibhatla, I., Gautam, D. K. and Mishra, R. K. (2019). SETDB1 modulates the differentiation of both the crystal cells and the lamellocytes in Drosophila. Dev Biol. PubMed ID: 31422102
Summary:
SETDB1 catalyzes the epigenetic mark of lysine-9 methylation of histone-3. This study explores the role of SETDB1 in Drosophila hematopoiesis. SETDB1 controls the differentiation of matured blood cells in wandering third instar larvae. There are three matured blood cells in wild type Drosophila melanogaster: plasmatocytes, crystal cells and lamellocytes. Loss-of-function mutants of SETDB1 show hematopoietic defects; increased blood cell proliferation, decreased number of crystal cells, greater differentiation of blood cells into lamellocytes, dysplasia of the anterior lobes of lymph gland and presence of hematopoietic tumors. Cell type specific knockdown of SETDB1 provided similar phenotype i.e., decreased number of crystal cells and an increase in lamellocyte differentiation. In animals with loss of function of SETDB1, Notch pathway was downregulated. Further, over-expression of SETDB1 in blood cells resulted in an increase in the number of crystal cells. This increase is accompanied with an increase in the number of Notch(ICD) expressing cells. Genetic rescue was performed using UAS-GAL4 system to rescue loss of function SETDB1 mutants. The data show that the rescued larvae carrying a wild type copy of SETDB1 in mutant background are devoid of blood tumors. A novel dual function of SETDB1 methylatransferase was identified as a critical regulator of two of the matured hemocytes, crystal cells and lamellocytes. A novel role of SETDB1 is proposed in modulating the differentiation of crystal cells and lamellocytes from a common progenitor and underscore the importance of SETDB1 in Drosophila blood tumor suppression.
Wilky, H., Chari, S., Govindan, J. and Amodeo, A. A. (2019). Histone concentration regulates the cell cycle and transcription in early development. Development. PubMed ID: 31511251
Summary:
The early embryos of many animals including flies, fish, and frogs have unusually rapid cell cycles and delayed onset of transcription. These divisions are dependent on maternally supplied RNAs and proteins including histones. Previous work suggests that the pool size of maternally provided histones can alter the timing of zygotic genome activation (ZGA) in frogs and fish. This study examine the effects of under and overexpression of maternal histones in Drosophila embryogenesis. Decreasing histone concentration advances zygotic transcription, cell cycle elongation, Chk1 activation, and gastrulation. Conversely, increasing histone concentration delays transcription and results in an additional nuclear cycle before gastrulation. Numerous zygotic transcripts are sensitive to histone concentration, and the promoters of histone sensitive genes are associated with specific chromatin features linked to increased histone turnover. These include enrichment of the pioneer transcription factor Zelda and lack of SIN3A and associated histone deacetylases. These findings uncover a critical regulatory role for histone concentrations in ZGA of Drosophila.
Ray, J., Munn, P. R., Vihervaara, A., Lewis, J. J., Ozer, A., Danko, C. G. and Lis, J. T. (2019). Chromatin conformation remains stable upon extensive transcriptional changes driven by heat shock. Proc Natl Acad Sci U S A. PubMed ID: 31506350
Summary:
Heat shock (HS) initiates rapid, extensive, and evolutionarily conserved changes in transcription that are accompanied by chromatin decondensation and nucleosome loss at HS loci. This study employed in situ Hi-C to determine how heat stress affects long-range chromatin conformation in human and Drosophila cells. Compartments and topologically associating domains (TADs) remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. Although most interactions between HSF1 binding sites and target promoters were established in the nonheat shock (NHS) condition, a subset increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the HSF1 bound sites (putative enhancers), and contact frequency with a target promoter accurately predicted which up-regulated genes were direct targets of HSF1 during HS. The results suggest that the chromatin conformation necessary for a robust HS response is preestablished in NHS cells of diverse metazoan species.
Molnar, C., Heinen, J. P., Reina, J., Llamazares, S., Palumbo, E., Breschi, A., Gay, M., Villarreal, L., Vilaseca, M., Pollarolo, G. and Gonzalez, C. (2019). The histone code reader PHD finger protein 7 controls sex-linked disparities in gene expression and malignancy in Drosophila. Sci Adv 5(8): eaaw7965. PubMed ID: 31453329
Summary:
The notable male predominance across many human cancer types remains unexplained. This study shows that Drosophila l(3)mbt brain tumors are more invasive and develop as malignant neoplasms more often in males than in females. By quantitative proteomics, a signature of proteins was identified that are differentially expressed between male and female tumor samples. Prominent among them is the conserved chromatin reader PHD finger protein 7 (Phf7). Phf7 depletion reduces sex-dependent differences in gene expression and suppresses the enhanced malignant traits of male tumors. These results identify potential regulators of sex-linked tumor dimorphism and show that these genes may serve as targets to suppress sex-linked malignant traits.
AlHaj Abed, J., Erceg, J., Goloborodko, A., Nguyen, S. C., McCole, R. B., Saylor, W., Fudenberg, G., Lajoie, B. R., Dekker, J., Mirny, L. A. and Wu, C. T. (2019). Highly structured homolog pairing reflects functional organization of the Drosophila genome. Nat Commun 10(1): 4485. PubMed ID: 31582763
Summary:
Trans-homolog interactions have been studied extensively in Drosophila, where homologs are paired in somatic cells and transvection is prevalent. Nevertheless, the detailed structure of pairing and its functional impact have not been thoroughly investigated. Accordingly, this study generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, showing that homologs pair with varying precision genome-wide, in addition to establishing trans-homolog domains and compartments. This study revealed at least two forms of pairing: tight pairing, spanning contiguous small domains, and loose pairing, consisting of single larger domains. Strikingly, active genomic regions (A-type compartments, active chromatin, expressed genes) correlated with tight pairing, suggesting that pairing has a functional implication genome-wide. Finally, using RNAi and haplotype-resolved Hi-C, it was shown that disruption of pairing-promoting factors results in global changes in pairing, including the disruption of some interaction peaks.

Monday, October 21st - Adult neural development and function

Chen, H. L., Stern, U. and Yang, C. H. (2019). Molecular control limiting sensitivity of sweet taste neurons in Drosophila. Proc Natl Acad Sci U S A 116(40): 20158-20168. PubMed ID: 31527261
Summary:
To assess the biological value of environmental stimuli, animals' sensory systems must accurately decode both the identities and the intensities of these stimuli. While much is known about the mechanism by which sensory neurons detect the identities of stimuli, less is known about the mechanism that controls how sensory neurons respond appropriately to different intensities of stimuli. The ionotropic receptor IR76b has been shown to be expressed in different Drosophila chemosensory neurons for sensing a variety of chemicals. This study show that IR76b plays an unexpected role in lowering the sensitivity of Drosophila sweet taste neurons. First, IR76b mutants exhibited clear behavioral responses to sucrose and acetic acid (AA) at concentrations that were too low to trigger observable behavioral responses from WT animals. Second, IR76b is expressed in many sweet neurons on the labellum, and these neurons responded to both sucrose and AA. Removing IR76b from the sweet neurons increased their neuronal responses as well as animals' behavioral responses to sucrose and AA. Conversely, overexpressing IR76b in the sweet neurons decreased their neuronal as well as animals' behavioral responses to sucrose and AA. Last, IR76b's response-lowering ability has specificity: IR76b mutants and WT showed comparable responses to capsaicin when the mammalian capsaicin receptor VR1 was ectopically expressed in their sweet neurons. These findings suggest that sensitivity of Drosophila sweet neurons to their endogenous ligands is actively limited by IR76b and uncover a potential molecular target by which contexts can modulate sensitivity of sweet neurons.
Scheuermann, E. A. and Smith, D. P. (2019). Odor-specific deactivation defects in a Drosophila odorant binding protein mutant. Genetics. PubMed ID: 31492805
Summary:
Insect odorant binding proteins are a large, diverse group of low molecular weight proteins secreted into the fluid bathing olfactory and gustatory neuron dendrites. The best-characterized OBP, LUSH (OBP76a) enhances pheromone sensitivity enabling detection of physiological levels of the male-specific pheromone, 11-cis vaccenyl acetate. The role of the other OBPs encoded in the Drosophila genome is largely unknown. Using CRISPR-Cas9 this study generated and characterized the loss of function phenotype for two genes encoding homologous OBPs, OS-E (OBP83b) and OS-F (OBP83a). Instead of activation defects, these extracellular proteins are required for normal deactivation of odorant responses to a subset of odorants. Remarkably, odorants detected by the same odorant receptor are differentially affected by the loss of the OBPs, revealing an odorant-specific role in deactivation kinetics. In stark contrast to lush mutants, the OS-E/F mutants have normal activation kinetics to the affected odorants, even at low stimulus concentrations, suggesting these OBPs are not competing for these ligands with the odorant receptors. It was also shown that OS-E and OS-F are functionally redundant as either is sufficient to revert the mutant phenotype in transgenic rescue experiments. These findings expand understanding of the roles of OBPs to include deactivation of odorant responses.
Siegenthaler, D., Escribano, B., Brauler, V. and Pielage, J. (2019). Selective suppression and recall of long-term memories in Drosophila. PLoS Biol 17(8): e3000400. PubMed ID: 31454345
Summary:
Adaptive decision-making depends on the formation of novel memories. In Drosophila, the mushroom body (MB) is the site of associative olfactory long-term memory (LTM) storage. However, due to the sparse and stochastic representation of olfactory information in Kenyon cells (KCs), genetic access to individual LTMs remains elusive. This study developed a cAMP response element (CRE)-activity-dependent memory engram label (CAMEL) tool that genetically tags KCs responding to the conditioned stimulus (CS). CAMEL activity depends on protein-synthesis-dependent aversive LTM conditioning and reflects the time course of CRE binding protein 2 (CREB2) activity during natural memory formation. Inhibition of LTM-induced CAMEL neurons reduces memory expression, and artificial optogenetic reactivation is sufficient to evoke aversive behavior phenocopying memory recall. Together, these data are consistent with CAMEL neurons marking a subset of engram KCs encoding individual memories. This study provides new insights into memory circuitry organization and an entry point towards cellular and molecular understanding of LTM storage.
Caipo, L., Gonzalez-Ramirez, M. C., Guzman-Palma, P., Contreras, E. G., Palominos, T., Fuenzalida-Uribe, N., Hassan, B. A., Campusano, J. M., Sierralta, J. and Oliva, C. (2019). Slit neuronal secretion coordinates optic lobe morphogenesis in Drosophila. Dev Biol. PubMed ID: 31606342
Summary:
Slit is an evolutionary conserved protein essential for the development of the nervous system. For signaling, Slit has to bind to its cognate receptor Robo, a single-pass transmembrane protein. Although the Slit/Robo signaling pathway is well known for its involvement in axon guidance, it has also been associated to boundary formation in the Drosophila visual system. In the optic lobe, Slit is expressed in glial cells, positioned at the boundaries between developing neuropils, and in neurons of the medulla ganglia. Although it has been assumed that glial cells provide Slit to the system, the contribution of the neuronal expression has not been tested. This study shows that, contrary to what was previously thought, Slit protein provided by medulla neurons is also required for boundary formation and morphogenesis of the optic lobe. Furthermore, tissue specific rescue using modified versions of Slit demonstrates that this protein acts at long range and does not require processing by extracellular proteases. These data shed new light on understanding of the cellular mechanisms involved in Slit function in the fly visual system morphogenesis.
Courgeon, M. and Desplan, C. (2019). Coordination between stochastic and deterministic specification in the Drosophila visual system. Science 366(6463). PubMed ID: 31582524
Summary:
Sensory systems use stochastic fate specification to increase their repertoire of neuronal types. How these stochastic decisions are coordinated with the development of their targets is unknown. In the Drosophila retina, two subtypes of ultraviolet-sensitive R7 photoreceptors are stochastically specified. In contrast, their targets in the brain are specified through a deterministic program. This study identified subtypes of the main target of R7, the Dm8 neurons, each specific to the different subtypes of R7s. Dm8 subtypes are produced in excess by distinct neuronal progenitors, independently from R7. After matching with their cognate R7, supernumerary Dm8s are eliminated by apoptosis. Two interacting cell adhesion molecules, Dpr11 and DIPgamma, are essential for the matching of one of the synaptic pairs. These mechanisms allow the qualitative and quantitative matching of R7 and Dm8 and thereby permit the stochastic choice made in R7 to propagate to the brain.
Sayin, S., De Backer, J. F., Siju, K. P., Wosniack, M. E., Lewis, L. P., Frisch, L. M., Gansen, B., Schlegel, P., Edmondson-Stait, A., Sharifi, N., Fisher, C. B., Calle-Schuler, S. A., Lauritzen, J. S., Bock, D. D., Costa, M., Jefferis, G., Gjorgjieva, J. and Grunwald Kadow, I. C. (2019). A neural circuit arbitrates between persistence and withdrawal in hungry Drosophila. Neuron. PubMed ID: 31471123
Summary:
In pursuit of food, hungry animals mobilize significant energy resources and overcome exhaustion and fear. How need and motivation control the decision to continue or change behavior is not understood. Using a single fly treadmill, this study shows that hungry flies persistently track a food odor and increase their effort over repeated trials in the absence of reward suggesting that need dominates negative experience. It was further shown that odor tracking is regulated by two mushroom body output neurons (MBONs) connecting the MB to the lateral horn. These MBONs, together with dopaminergic neurons and Dop1R2 signaling, control behavioral persistence. Conversely, an octopaminergic neuron, VPM4, which directly innervates one of the MBONs, acts as a brake on odor tracking by connecting feeding and olfaction. Together, these data suggest a function for the MB in internal state-dependent expression of behavior that can be suppressed by external inputs conveying a competing behavioral drive.

Thursday, October 17th - Gonadogenesis

Tanabe, K., Awane, R., Shoda, T., Yamazoe, K. and Inoue, Y. H. (2019). Mutations in mxc tumor-suppressor gene induce chromosome instability in Drosophila male meiosis. Cell Struct Funct. PubMed ID: 31484839
Summary:
Drosophila Mxc protein is a component of the histone locus body (HLB), which is required for the expression of canonical histone genes. A fraction of the spermatids in hypomorphic mxcG46 mutants contained extra micronuclei or abnormally sized nuclei. Lagging chromosomes were observed to retain between chromosomal masses separated toward spindle poles at telophase I. Time-lapse recordings show that micronuclei were generated from lagging chromosomes, and the abnormal chromosomes in mxcG46 mutants lacked centromeres. In normal spermatocyte nuclei, the histone locus body component FLASH, a protein that plays an essential role in 3' end processing of replication-dependent histone pre-mRNAs, colocalized with Mxc, whereas FLASH was dispersed in mxcG46 spermatocyte nuclei. Furthermore, genetic interactions were observed between Mxc and other histone locus body (HLB) components in meiotic chromosome segregation, suggesting that inhibition of HLB formation is responsible for aberrant chromosome segregation in mxcG46. Quantitative real-time PCR revealed that canonical histone mRNA levels were decreased in mxcG46. Lastly, similar meiotic phenotypes appeared in the spermatids of histone H4 mutants and in the spermatids in testes depleted for chromosome-construction factors. Considering these genetic data, it is proposed that abnormal chromosome segregation leading to CIN development results from a loss of chromosome integrity caused by diminished canonical histone levels in mxc mutants.
Park, J. H., Nguyen, T. T. N., Lee, E. M., Castro-Aceituno, V., Wagle, R., Lee, K. S., Choi, J. and Song, Y. H. (2019). Role of p53 isoforms in the DNA damage response during Drosophila oogenesis. Sci Rep 9(1): 11473. PubMed ID: 31391501
Summary:
The tumor suppressor p53 is involved in the DNA damage response and induces cell cycle arrest or apoptosis upon DNA damage. Drosophila p53 encodes two isoforms, p53A and p53B, that induce apoptosis in somatic cells. To investigate the roles of Drosophila p53 isoforms in female germline cells, the DNA damage response was analyzed in the adult ovary. Early oogenesis was sensitive to irradiation and lok-, p53-, and hid-dependent cell death occurred rapidly after both low- and high-dose irradiation. Both p53 isoforms were responsible for this cell death. On the other hand, delayed cell death in mid-oogenesis was induced at a low level only after high-dose irradiation in a p53-independent manner. The daily egg production, which did not change after low-dose irradiation, was severely reduced after high-dose irradiation in p53 mutant females due to the loss of germline stem cells. When the p53A or p53B isoforms were expressed in the germline cells in the p53 mutant females at levels that do not affect normal oogenesis, p53A, but not p53B, restored the fertility of the irradiated female. In summary, moderate expression of p53A is critical to maintain the function of germline stem cells during normal oogenesis as well as after high-dose irradiation.
Weaver, L. N. and Drummond-Barbosa, D. (2019). The nuclear receptor seven up functions in adipocytes and oenocytes to control distinct steps of Drosophila oogenesis. Dev Biol. PubMed ID: 31470019
Summary:
>Reproduction is intimately linked to the physiology of an organism. Nuclear receptors are widely expressed transcription factors that mediate the effects of many circulating molecules on physiology and reproduction. While multiple studies have focused on the roles of nuclear receptors intrinsically in the ovary, it remains largely unknown how the actions of nuclear receptors in peripheral tissues influence oogenesis. This study identified the nuclear receptor encoded by svp as a novel regulator of oogenesis in adult Drosophila. Global somatic knockdown of svp reduces egg production by increasing GSC loss, death of early germline cysts, and degeneration of vitellogenic follicles. Tissue-specific knockdown experiments revealed that svp remotely controls these different steps of oogenesis through separate mechanisms involving distinct tissues. Specifically, adipocyte-specific svp knockdown impairs GSC maintenance and early germline cyst survival, whereas oenocyte-specific svp knockdown increases the death of vitellogenic follicles without any effects on GSCs or early cysts. These results illustrate that nuclear receptors can control reproduction through a variety of mechanisms involving peripheral tissues.
Steinhauer, J., Statman, B., Fagan, J. K., Borck, J., Surabhi, S., Yarikipati, P., Edelman, D. and Jenny, A. (2019). Combover interacts with the axonemal component Rsp3 and is required for Drosophila sperm individualization. Development 146(17). PubMed ID: 31391193
Summary:
Gamete formation is key to survival of higher organisms. In male animals, spermatogenesis gives rise to interconnected spermatids that differentiate and individualize into mature sperm, each tightly enclosed by a plasma membrane. In Drosophila melanogaster, individualization of sister spermatids requires the formation of specialized actin cones that synchronously move along the sperm tails, removing inter-spermatid bridges and most of the cytoplasm. This study shows that Combover (Cmb), originally identified as an effector of planar cell polarity (PCP) under control of Rho kinase, is essential for sperm individualization. cmb mutants are male sterile, with actin cones that fail to move in a synchronized manner along the flagella, despite being correctly formed and polarized initially. These defects are germline autonomous, independent of PCP genes, and can be rescued by wild-type Cmb, but not by a version of Cmb in which known Rho kinase phosphorylation sites are mutated. Furthermore, Cmb binds to the axonemal component Radial spoke protein 3, knockdown of which causes similar individualization defects, suggesting that Cmb coordinates the individualization machinery with the microtubular axonemes.
Witt, E., Benjamin, S., Svetec, N. and Zhao, L. (2019). Testis single-cell RNA-seq reveals the dynamics of de novo gene transcription and germline mutational bias in Drosophila. Elife 8. PubMed ID: 31418408
Summary:
The testis is a peculiar tissue in many respects. It shows patterns of rapid gene evolution and provides a hotspot for the origination of genetic novelties such as de novo genes, duplications and mutations. To investigate the expression patterns of genetic novelties across cell types, single-cell RNA-sequencing was performed of adult Drosophila testis. New genes were expressed in various cell types, the patterns of which may be influenced by their mode of origination. In particular, lineage-specific de novo genes are commonly expressed in early spermatocytes, while young duplicated genes are often bimodally expressed. Analysis of germline substitutions suggests that spermatogenesis is a highly reparative process, with the mutational load of germ cells decreasing as spermatogenesis progresses. By elucidating the distribution of genetic novelties across spermatogenesis, this study provides a deeper understanding of how the testis maintains its core reproductive function while being a hotbed of evolutionary innovation.
Ng, C. L., Qian, Y. and Schulz, C. (2019). Notch and Delta are required for survival of the germline stem cell lineage in testes of Drosophila melanogaster. PLoS One 14(9): e0222471. PubMed ID: 31513679
Summary:
In all metazoan species, sperm is produced from germline stem cells. These self-renew and produce daughter cells that amplify and differentiate dependent on interactions with somatic support cells. In the male gonad of Drosophila melanogaster, the germline and somatic cyst cells co-differentiate as cysts, an arrangement in which the germline is completely enclosed by cytoplasmic extensions from the cyst cells. Notch is a developmentally relevant receptor in a pathway requiring immediate proximity with the signal sending cell. This study shows that Notch is expressed in the cyst cells of wild-type testes. Notch becomes activated in the transition zone, an apical area of the testes in which the cyst cells express stage-specific transcription factors and the enclosed germline finalizes transit-amplifying divisions. Reducing the ligand Delta from the germline cells via RNA-Interference or reducing the receptor Notch from the cyst cells via CRISPR resulted in cell death concomitant with loss of germline cells from the transition zone. This shows that Notch signaling is essential for the survival of the germline stem cell lineage.

Wednesday, October 16th - Synapse and Vesicles

Weber, T., Stephan, R., Moreno, E. and Pielage, J. (2019). The ankyrin repeat domain controls presynaptic localization of Drosophila Ankyrin2 and is essential for synaptic stability. Front Cell Dev Biol 7: 148. PubMed ID: 31475145
Summary:
The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. This study addresses the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. A P[acman] based rescue approach was used to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. Specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, these data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance.
Vasin, A., Sabeva, N., Torres, C., Phan, S., Bushong, E. A., Ellisman, M. H. and Bykhovskaia, M. (2019). Two pathways for the activity-dependent growth and differentiation of synaptic boutons in Drosophila. eNeuro 6(4). PubMed ID: 31387877
Summary:
Synapse formation can be promoted by intense activity. At the Drosophila larval neuromuscular junction (NMJ), new synaptic boutons can grow acutely in response to patterned stimulation. This study combined confocal imaging with electron microscopy and tomography to investigate the initial stages of growth and differentiation of new presynaptic boutons at the Drosophila NMJ. The new boutons can form rapidly in intact larva in response to intense crawling activity, and two different patterns of bouton formation and maturation were observed. The first pathway involves the growth of filopodia followed by a formation of boutons that are initially devoid of synaptic vesicles (SVs) but filled with filamentous matrix. The second pathway involves rapid budding of synaptic boutons packed with SVs, and these more mature boutons are sometimes capable of exocytosis/endocytosis. Intense activity predominantly promotes the second pathway, i.e., budding of more mature boutons filled with SVs. This pathway depends on Synapsin (Syn), a neuronal protein which reversibly associates with SVs and mediates their clustering via a protein kinase A (PKA)-dependent mechanism. Finally, advantage of the temperature-sensitive mutant sei to demonstrate that seizure activity can promote very rapid budding of new boutons filled with SVs, and this process occurs at scale of minutes. Altogether, these results demonstrate that intense activity acutely and selectively promotes rapid budding of new relatively mature presynaptic boutons filled with SVs, and that this process is regulated via a PKA/Syn-dependent pathway.
Ly, S., Strus, E. and Naidoo, N. (2019). Genetic disruption of the putative binding site for Homer on DmGluRA reduces sleep in Drosophila. Sleep. PubMed ID: 31418019
Summary:
Homer proteins mediate plasticity and signaling at the postsynaptic density of neurons and are necessary for sleep and synaptic remodeling during sleep. The goal of this study was to investigate the mechanisms of sleep regulation by Homer signaling. Using the Drosophila animal model, this study demonstrates that knockdown of Homer specifically in the brain reduces sleep and that Drosophila Homer binds to the sole Drosophila mGluR, known as DmGluRA. This is the first evidence that DmGluRA (metabotropic Glutamate Receptor), which bears greatest homology to group II mammalian mGluRs, shares functional homology with group I mGluRs which couple to Homer proteins in mammals. Since sleep is associated with the physical dissociation of Homer and mGluRs proteins at the synapse attempts were made to determine the functional necessity of Homer and DmGluRA interaction in sleep regulation. Using the CRISPR/Cas9 gene editing system, a targeted amino acid replacement of the putative binding site for Homer on DmGluRA was generated to prevent Homer and DmGluRA protein binding. Loss of the conserved proline-rich PPXXF sequence on DmGluRA reduces Homer/DmGluRA associations and significantly reduces sleep amount. Thus, this study identified a conserved mechanism of synaptic plasticity in Drosophila and has demonstrated that the interaction of Homer with DmGluRA is necessary to promote sleep.
Mezzofanti, E., Ignesti, M., Hsu, T., Gargiulo, G. and Cavaliere, V. (2019). Vps28 is involved in the intracellular trafficking of Awd, the Drosophila homolog of NME1/2. Front Physiol 10: 983. PubMed ID: 31427986
Summary:
The Awd (abnormal wing discs) gene is the Drosophila homolog of human NME1 and NME2 metastasis suppressor genes. These genes play a key role in tumor progression. Extensive studies revealed that intracellular NME1/2 protein levels could be related to either favorable or poor prognosis depending on tissue context. More recently, extracellular activities of NME1/2 proteins have also been reported, including a tumor- promoting function. This study used Drosophila as a genetic model to investigate the mechanism controlling intra- and extracellular levels of NME1/2. The role was examined of several components of the ESCRT (endosomal sorting complex required for transport) complex in controlling Awd trafficking. The Vps28 component of the ESCRT-I complex is required for maintenance of normal intracellular level of Awd in larval adipocytes. Blocking of Shibire (Shi)/Dynamin function strongly- lowers Awd intracellular level. To further investigate this down regulative effect, the distribution of endosomal markers was analyzed in wild type and Shi-defective adipocytes. The results suggest that Awd does not enter CD63-positive endosomes. Interestingly, it was found that, in fat body cells, Awd partly colocalizes with the ESCRT accessory component ALiX, the ALG-2 (apoptosis-linked gene 2)-interacting protein X. Moreover, the intracellular levels of both proteins are downregulated by blocking the function of the Dynamin encoded by the shibire gene.
Ojelade, S. A., Lee, T. V., Giagtzoglou, N., Yu, L., Ugur, B., Li, Y., Duraine, L., Zuo, Z., Petyuk, V., De Jager, P. L., Bennett, D. A., Arenkiel, B. R., Bellen, H. J. and Shulman, J. M. (2019). cindr, the Drosophila homolog of the CD2AP Alzheimer's disease risk gene, is required for synaptic transmission and proteostasis. Cell Rep 28(7): 1799-1813 PubMed ID: 31412248
Summary:
The Alzheimer's disease (AD) susceptibility gene, CD2-associated protein (CD2AP), encodes an actin binding adaptor protein, but its function in the nervous system is largely unknown. Loss of the Drosophila ortholog cindr enhances neurotoxicity of human Tau, which forms neurofibrillary tangle pathology in AD. Cindr is expressed in neurons and present at synaptic terminals. cindr mutants show impairments in synapse maturation and both synaptic vesicle recycling and release. Cindr associates and genetically interacts with 14-3-3zeta, regulates the ubiquitin-proteasome system, and affects turnover of Synapsin and the plasma membrane calcium ATPase (PMCA). Loss of cindr elevates PMCA levels and reduces cytosolic calcium. Studies of Cd2ap null mice support a conserved role in synaptic proteostasis, and CD2AP protein levels are inversely related to Synapsin abundance in human postmortem brains. These results reveal CD2AP neuronal requirements with relevance to AD susceptibility, including for proteostasis, calcium handling, and synaptic structure and function.
Tenedini, F. M., Saez Gonzalez, M., Hu, C., Pedersen, L. H., Petruzzi, M. M., Spitzweck, B., Wang, D., Richter, M., Petersen, M., Szpotowicz, E., Schweizer, M., Sigrist, S. J., Calderon de Anda, F. and Soba, P. (2019). Maintenance of cell type-specific connectivity and circuit function requires Tao kinase. Nat Commun 10(1): 3506. PubMed ID: 31383864
Summary:
Sensory circuits are typically established during early development, yet how circuit specificity and function are maintained during organismal growth has not been elucidated. To gain insight this study quantitatively investigated synaptic growth and connectivity in the Drosophila nociceptive network during larval development. Cnnectivity between primary nociceptors and their downstream neurons was shown to scale with animal size. The conserved Ste20-like kinase Tao was identified as a negative regulator of synaptic growth required for maintenance of circuit specificity and connectivity. Loss of Tao kinase resulted in exuberant postsynaptic specializations and aberrant connectivity during larval growth. Using functional imaging and behavioral analysis, loss of Tao-induced ectopic synapses with inappropriate partner neurons were shown to be functional and altered behavioral responses in a connection-specific manner. These data show that fine-tuning of synaptic growth by Tao kinase is required for maintaining specificity and behavioral output of the neuronal network during animal growth.

Tuesday, October 15th - Signal Transduction

Ugrankar, R., Bowerman, J., Hariri, H., Chandra, M., Chen, K., Bossanyi, M. F., Datta, S., Rogers, S., Eckert, K. M., Vale, G., Victoria, A., Fresquez, J., McDonald, J. G., Jean, S., Collins, B. M. and Henne, W. M. (2019). Drosophila Snazarus regulates a lipid droplet population at plasma membrane-droplet contacts in adipocytes. Dev Cell 50(5): 557-572. PubMed ID: 31422916
Summary:
Adipocytes store nutrients as lipid droplets (LDs), but how they organize their LD stores to balance lipid uptake, storage, and mobilization remains poorly understood. Using Drosophila fat body (FB) adipocytes, this study characterized spatially distinct LD populations that are maintained by different lipid pools. Peripheral LDs (pLDs) were identified that make close contact with the plasma membrane (PM) and are maintained by lipophorin-dependent lipid trafficking. pLDs are distinct from larger cytoplasmic medial LDs (mLDs), which are maintained by FASN1-dependent de novo lipogenesis. Sorting nexin CG1514 or Snazarus (Snz) associates with pLDs and regulates LD homeostasis at ER-PM contact sites. Loss of SNZ perturbs pLD organization, whereas Snz over-expression drives LD expansion, triacylglyceride production, starvation resistance, and lifespan extension through a DESAT1-dependent pathway. It is proposed that Drosophila adipocytes maintain spatially distinct LD populations and identify Snz as a regulator of LD organization and inter-organelle crosstalk.
Nunes de Almeida, F., Walther, R. F., Presse, M. T., Vlassaks, E. and Pichaud, F. (2019). Cdc42 defines apical identity and regulates epithelial morphogenesis by promoting apical recruitment of Par6-aPKC and Crumbs. Development 146(15). PubMed ID: 31405903
Summary:
Cdc42 regulates epithelial morphogenesis together with the Par complex (Baz/Par3-Par6-aPKC), Crumbs (Crb/CRB3) and Stardust (Sdt/PALS1). However, how these proteins work together and interact during epithelial morphogenesis is not well understood. To address this issue, this study used the genetically amenable Drosophila pupal photoreceptor and follicular epithelium. During epithelial morphogenesis active Cdc42 accumulates at the developing apical membrane and cell-cell contacts, independently of the Par complex and Crb. However, membrane localization of Baz, Par6-aPKC and Crb all depend on Cdc42. Although binding of Cdc42 to Par6 is not essential for the recruitment of Par6 and aPKC to the membrane, it is required for their apical localization and accumulation, which was found to also depend on Par6 retention by Crb. In the pupal photoreceptor, membrane recruitment of Par6-aPKC also depends on Baz. This work shows that Cdc42 is required for this recruitment and suggests that this factor promotes the handover of Par6-aPKC from Baz onto Crb. Altogether, it is proposed that Cdc42 drives morphogenesis by conferring apical identity, Par-complex assembly and apical accumulation of Crb.
Rozbesky, D., Robinson, R. A., Jain, V., Renner, M., Malinauskas, T., Harlos, K., Siebold, C. and Jones, E. Y. (2019). Diversity of oligomerization in Drosophila semaphorins suggests a mechanism of functional fine-tuning. Nat Commun 10(1): 3691. PubMed ID: 31417095
Summary:
Semaphorin ligands and their plexin receptors are one of the major cell guidance factors that trigger localised changes in the cytoskeleton. Binding of semaphorin homodimer to plexin brings two plexins in close proximity which is a prerequisite for plexin signalling. This model appears to be too simplistic to explain the complexity and functional versatility of these molecules. This study determine crystal structures for all members of Drosophila class 1 and 2 semaphorins. Unlike previously reported semaphorin structures, Sema1a, Sema2a and Sema2b show stabilisation of sema domain dimer formation via a disulfide bond. Unexpectedly, structural and biophysical data show Sema1b is a monomer suggesting that semaphorin function may not be restricted to dimers. Semaphorins can form heterodimers with members of the same semaphorin class. This heterodimerization provides a potential mechanism for cross-talk between different plexins and co-receptors to allow fine-tuning of cell signalling.
Suzawa, M., Muhammad, N. M., Joseph, B. S. and Bland, M. L. (2019). The Toll signaling pathway targets the insulin-like peptide Dilp6 to inhibit growth in Drosophila. Cell Rep 28(6): 1439-1446. PubMed ID: 31390559
Summary:
Chronic enteropathogen infection in early childhood reduces circulating insulin-like growth factor 1 (IGF1) levels and restricts growth. Pathogen-derived molecules activate host Toll-like receptors to initiate the immune response, but whether this pathway contributes to growth inhibition is unclear. In Drosophila, activation of Toll receptors in larval fat body suppresses whole-animal growth. In this study, using a transcriptomic approach, identified Drosophila insulin-like peptide 6 (Dilp6), a fat-body-derived IGF1 ortholog, as a selective target of Toll signaling induced by infection or genetic activation of the pathway. Using a tagged allele that was generated to measure endogenous Dilp6, a marked reduction in circulating hormone levels was found. Restoring Dilp6 expression in fat body rescues growth in animals with active Toll signaling. These results establish that Toll signaling reduces growth by inducing hormone insufficiency, implying a mechanistic link between innate immune signaling and endocrine regulation of growth.
Thuveson, M., Gaengel, K., Collu, G. M., Chin, M. L., Singh, J. and Mlodzik, M. (2019). Integrins are required for synchronous ommatidial rotation in the Drosophila eye linking planar cell polarity signalling to the extracellular matrix. Open Biol 9(8): 190148. PubMed ID: 31409231
Summary:
Integrins mediate the anchorage between cells and their environment, the extracellular matrix (ECM), and form transmembrane links between the ECM and the cytoskeleton, a conserved feature throughout development and morphogenesis of epithelial organs. This study demonstrates that integrins and components of the ECM are required during the planar cell polarity (PCP) signalling-regulated cell movement of ommatidial rotation in the Drosophila eye. The loss-of-function mutations of integrins or ECM components cause defects in rotation, with mutant clusters rotating asynchronously compared to wild-type clusters. Initially, mutant clusters tend to rotate faster, and at later stages they fail to be synchronous with their neighbours, leading to aberrant rotation angles and resulting in a disorganized ommatidial arrangement in adult eyes. It was further demonstrated that integrin localization changes dynamically during the rotation process. The data suggest that core Frizzled/PCP factors, acting through RhoA and Rho kinase, regulate the function/activity of integrins and that integrins thus contribute to the complex interaction network of PCP signalling, cell adhesion and cytoskeletal elements required for a precise and synchronous 90 degrees rotation movement.
Tsai, C. R. and Galko, M. J. (2019). Casein kinase 1alpha decreases beta-catenin levels at adherens junctions to facilitate wound closure in Drosophila larvae. Development. PubMed ID: 31511254
Summary:
Skin wound repair is essential to restore barrier function and prevent infection after tissue damage. Wound-edge epidermal cells migrate as a sheet to close the wound. However, it is still unclear how cell-cell junctions are regulated during wound closure (WC). To study this, adherens junctions were examined during WC in Drosophila larvae. beta-catenin is reduced at the lateral cell-cell junctions of wound-edge epidermal cells in the early healing stages. Destruction complex components, including Ck1alpha, GSK3beta and beta-TrCP suppress beta-catenin levels in the larval epidermis. Tissue-specific RNAi targeting these genes also caused severe WC defects. The Ck1alpha (RNAi) -induced WC defect is related to adherens junctions because loss of either beta-catenin or E-cadherin significantly rescued this WC defect. In contrast, TCF (RNAi) does not rescue the Ck1alpha (RNAi) -induced WC defect, suggesting that Wnt signaling is not related to this defect. Direct overexpression of beta-catenin recapitulates most of the features of Ck1alpha reduction during wounding. Finally, loss of Ck1alpha also blocked junctional E-cadherin reduction around the wound. These results suggest that Ck1alpha and the destruction complex locally regulate cell adhesion to facilitate efficient wound repair.

Monday, October 14th - Adult Physiology

Kristensen, T. N., Loeschcke, V., Tan, Q., Pertoldi, C. and Mengel-From, J. (2019). Sex and age specific reduction in stress resistance and mitochondrial DNA copy number in Drosophila melanogaster. Sci Rep 9(1): 12305. PubMed ID: 31444377
Summary:
Environmental stresses such as extreme temperatures, dehydration and food deprivation may have distinct consequences for different age-classes and for males and females across species. This study investigated a natural population of the model organism Drosophila melanogaster. Males and females at ages 3, 19 and 35 days were tested for stress resistance; i.e. the ability of flies to cope with starvation and both cold and hot temperatures. Further, a measure of metabolic efficiency, namely mitochondrial DNA copy number (mtDNA CN), was tested in both sexes at all three age-classes. It was hypothesized that stress resistance is reduced at old age and more so in males, and that mtDNA CN is a biomarker for sex- and age-dependent reductions in the ability to cope with harsh environments. This study showed that: (1) males exhibit reduced starvation tolerance at old age, whereas older females are better in coping with periods without food compared to younger females, (2) heat tolerance decreases with increasing age in males but not in females, (3) cold tolerance is reduced at old age in both sexes, and (4) old males have reduced mtDNA CN whereas mtDNA CN slightly increases with age in females. In conclusion, these data provide strong evidence for trait and sex specific consequences of aging with females generally being better at coping with environmental stress at old age. The reduced mtDNA CN in old males suggests reduced metabolic efficiency and this may partly explain why males are less stress tolerant at old age than females. It is suggested that mtDNA CN might be a suitable biomarker for physiological robustness. These findings likely extend to other taxa than Drosophila and therefore the observations are discussed in relation to aging and sex specific lifespan across species.
Mossman, J. A., Biancani, L. M. and Rand, D. M. (2019). Mitochondrial genomic variation drives differential nuclear gene expression in discrete regions of Drosophila gene and protein interaction networks. BMC Genomics 20(1): 691. PubMed ID: 31477008
Summary:
Mitochondria perform many key roles in their eukaryotic hosts, from integrating signaling pathways through to modulating whole organism phenotypes. The > 1 billion years of nuclear and mitochondrial gene co-evolution has necessitated coordinated expression of gene products from both genomes that maintain mitochondrial, and more generally, eukaryotic cellular function. How mitochondrial DNA (mtDNA) variation modifies host fitness has proved a challenging question but has profound implications for evolutionary and medical genetics. In Drosophila, previous work has shown that recently diverged mtDNA haplotypes within-species can have more impact on organismal phenotypes than older, deeply diverged haplotypes from different species. This study tested the effects of mtDNA haplotype variation on gene expression in Drosophila under standardized conditions. Using the Drosophila Genetic Reference Panel (DGRP), a panel of mitonuclear genotypes was constructed that consists of factorial variation in nuclear and mtDNA genomes, with mtDNAs originating in D. melanogaster (2x haplotypes) and D. simulans (2x haplotypes).MtDNA haplotype variation unequivocally altered nuclear gene expression in both females and males, and mitonuclear interactions are pervasive modifying factors for gene expression. There was appreciable overlap between the sexes for mtDNA-sensitive genes, and considerable transcriptional variation attributed to particular mtDNA contrasts. These genes are generally found in low-connectivity gene co-expression networks, occur in gene clusters along chromosomes, are often flanked by non-coding RNA, and are under-represented among housekeeping genes. Finally, the giant (gt) transcription factor motif was identified as a putative regulatory sequence associated with mtDNA-sensitive genes. It is concluded that there are predictive conditions for nuclear genes that are influenced by mtDNA variation.
Ma, D., Bou-Sleiman, M., Joncour, P., Indelicato, C. E., Frochaux, M., Braman, V., Litovchenko, M., Storelli, G., Deplancke, B. and Leulier, F. (2019). Commensal gut bacteria buffer the impact of host genetic variants on Drosophila developmental traits under nutritional stress. iScience 19: 436-447. PubMed ID: 31422284
Summary:
Eukaryotic genomes encode several buffering mechanisms that robustly maintain invariant phenotypic outcome despite fluctuating environmental conditions. This study show that the Drosophila gut-associated commensals, represented by a single facultative symbiont, Lactobacillus plantarum (Lp(WJL)), constitutes a so far unexpected buffer that masks the contribution of the host's cryptic genetic variation (CGV) to developmental traits while the host is under nutritional stress. During chronic under-nutrition, Lp(WJL) consistently reduces variation in different host phenotypic traits and ensures robust organ patterning during development; Lp(WJL) also decreases genotype-dependent expression variation, particularly for development-associated genes. Evidence is provided that Lp(WJL) buffers via reactive oxygen species (ROS) signaling whose inhibition impairs microbiota-mediated phenotypic robustness. This study thus identified a hitherto unappreciated contribution of the gut facultative symbionts to host fitness that, beyond supporting growth rates and maturation timing, confers developmental robustness and phenotypic homogeneity in times of nutritional stress.
Seong, K. M., Coates, B. S. and Pittendrigh, B. R. (2019). Cytochrome P450s Cyp4p1 and Cyp4p2 associated with the DDT tolerance in the Drosophila melanogaster strain 91-R. Pestic Biochem Physiol 159: 136-143. PubMed ID: 31400775
Summary:
Cytochrome P450s are part of a super-gene family that has undergone gene duplication, divergence, over-expression and, in some cases, loss of function. One such case is the 91-R and 91-C strains of common origin, in Drosophila melanogaster, whereby 91-R (DDT resistant strain) overexpresses Cyp4p1 and Cyp4p2 and both genes are lost in 91-C (DDT susceptible strain). This study used a comparative approach to demonstrate that transcription of Cyp4p1 and Cyp4p2 were constitutively up-regulated in the Drosophila melanogaster strain 91-R as compared to another DDT susceptible strain Canton-S which does not have a loss of function of these genes. Furthermore, significantly increased expression of Cyp4p1 and Cyp4p2 was induced in 91-R in response to sublethal DDT exposure, however, such induction did not occur in the DDT treated Canton-S. Additionally, fixed nucleotide variation within putative transcription factor binding sites of Cyp4p1 and Cyp4p2 promoters were observed between 91-R and Canton-S, however, their impact on transcription remains to be determined. Two GAL4/UAS transgenic strains with integrated heat shock-inducible Cyp4p1- or Cyp4p2-RNAi constructs within wild-type genetic backgrounds were developed. Following heat shock induction of Cyp4p1 and Cyp4p2 knockdown, these transgenic lines showed increased DDT mortality as compared to their corresponding non-heat shock controls. These results provide a functional link of Cyp4p1 and Cyp4p2 in conferring tolerance to DDT exposure.
Hudry, B., de Goeij, E., Mineo, A., Gaspar, P., Hadjieconomou, D., Studd, C., Mokochinski, J. B., Kramer, H. B., Placais, P. Y., Preat, T. and Miguel-Aliaga, I. (2019). Sex Differences in intestinal carbohydrate metabolism promote food intake and sperm maturation. Cell 178(4): 901-918.e916. PubMed ID: 31398343
Summary:
Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. This study used the intestine of Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. Carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signaling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this male-biased intestinal metabolic state in controlling food intake and sperm production through gut-derived citrate. This work uncovers a male gonad-gut axis coupling diet and sperm production, revealing that metabolic communication across organs is physiologically important. The instructive role of citrate in inter-organ communication might be significant in more biological contexts than previously recognized.
Lim, S., Jung, J., Yunusbaev, U., Ilyasov, R. and Kwon, H. W. (2019). Characterization and its implication of a novel taste receptor detecting nutrients in the honey bee, Apis mellifera. Sci Rep 9(1): 11620. PubMed ID: 31406120
Summary:
Umami taste perception indicates the presence of amino acids, which are essential nutrients. Although the physiology of umami perception has been described in mammals, how insects detect amino acids remains unknown except in Drosophila melanogaster. This study functionally characterized a gustatory receptor responding to L-amino acids in the western honey bee, Apis mellifera. Using a calcium-imaging assay and two-voltage clamp recording, it was found that one of the honey bee's gustatory receptors, AmGr10, functions as a broadly tuned amino acid receptor responding to glutamate, aspartate, asparagine, arginine, lysine, and glutamine, but not to other sweet or bitter compounds. Furthermore, the sensitivity of AmGr10 to these L-amino acids was dramatically enhanced by purine ribonucleotides, like inosine-5'-monophosphate (IMP). Contact sensory hairs in the mouthpart of the honey bee responded strongly to glutamate and aspartate, which house gustatory receptor neurons expressing AmGr10. Interestingly, AmGr10 protein is highly conserved among hymenopterans but not other insects, implying unique functions in eusocial insects.

Friday, October 12th - Cytoskeleton and Junctions

Mishra, A. K., Mondo, J. A., Campanale, J. P. and Montell, D. J. (2019). Coordination of protrusion dynamics within and between collectively migrating border cells by myosin II. Mol Biol Cell 30(19): 2490-2502. PubMed ID: 31390285
Summary:
Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell-cell adhesions and coordinate direction-sensing as they move. While nonmuscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. This study used genetics, Airyscan microscopy, live imaging, optogenetics, and Forster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. Myosin was found to accumulate transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin colocalize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo.
Persico, V., Callaini, G. and Riparbelli, M. G. (2019). The microtubule-depolymerizing Kinesin-13 Klp10A is enriched in the transition zone of the ciliary structures of Drosophila melanogaster. Front Cell Dev Biol 7: 173. PubMed ID: 31497602
Summary:
The precursor of the flagellar axoneme is already present in the primary spermatocytes of Drosophila melanogaster. During spermatogenesis each primary spermatocyte shows a centriole pair that moves to the cell membrane and organizes an axoneme-based structure, the cilium-like region (CLR). The CLRs persist through the meiotic divisions and are inherited by young spermatids. During spermatid differentiation the ciliary caps elongate giving rise to the sperm axoneme. Mutations in Klp10A, a kinesin-13 of Drosophila, results in defects of centriole/CLR organization in spermatocytes and of ciliary cap assembly in elongating spermatids. Reduced Klp10A expression also results in strong structural defects of sensory type I neurons. This study shows that this protein displays a peculiar localization during male gametogenesis. The Klp10A signal is first detected at the distal ends of the centrioles when they dock to the plasma membrane of young primary spermatocytes. At the onset of the first meiotic prometaphase, when the CLRs reach their full size, Klp10A is enriched in a distinct narrow area at the distal end of the centrioles and persists in elongating spermatids at the base of the ciliary cap. It is concluded that Klp10A could be a core component of the ciliary transition zone in Drosophila.
Lim, H. Y., Bao, H., Liu, Y. and Wang, W. (2019). Select septate junction proteins direct ROS-mediated paracrine regulation of Drosophila cardiac function. Cell Rep 28(6): 1455-1470. PubMed ID: 31390561
Summary:
Septate junction (SJ) complex proteins act in unison to provide a paracellular barrier and maintain structural integrity. This study has identified a non-barrier role of two individual SJ proteins, Coracle (Cora) and Kune-kune (Kune). Reactive oxygen species (ROS)-p38 MAPK signaling in non-myocytic pericardial cells (PCs) is important for maintaining normal cardiac physiology in Drosophila. However, the underlying mechanisms remain unknown. This study has found that in PCs, Cora and Kune are altered in abundance in response to manipulations of ROS-p38 signaling. Genetic analyses establish Cora and Kune as key effectors of ROS-p38 signaling in PCs on proper heart function. It was further determined that Cora regulates normal Kune levels in PCs, which in turn modulates normal Kune levels in the cardiomyocytes essential for proper heart function. These results thereby reveal select SJ proteins Cora and Kune as signaling mediators of the PC-derived ROS regulation of cardiac physiology.
Plutoni, C., Keil, S., Zeledon, C., Delsin, L. E. A., Decelle, B., Roux, P. P., Carreno, S. and Emery, G. (2019). Misshapen coordinates protrusion restriction and actomyosin contractility during collective cell migration. Nat Commun 10(1): 3940. PubMed ID: 31477736
Summary:
Collective cell migration is involved in development, wound healing and metastasis. In the Drosophila ovary, border cells (BC) form a small cluster that migrates collectively through the egg chamber. To achieve directed motility, the BC cluster coordinates the formation of protrusions in its leader cell and contractility at the rear. Restricting protrusions to leader cells requires the actin and plasma membrane linker Moesin. This study shows that the Ste20-like kinase Misshapen phosphorylates Moesin in vitro and in BC. Depletion of Misshapen disrupts protrusion restriction, thereby allowing other cells within the cluster to protrude. In addition, this study shows that Misshapen is critical to generate contractile forces both at the rear of the cluster and at the base of protrusions. Together, these results indicate that Misshapen is a key regulator of BC migration as it coordinates two independent pathways that restrict protrusion formation to the leader cells and induces contractile forces.
Ramanathan, S. P., Krajnc, M. and Gibson, M. C. (2019). Cell-size pleomorphism drives aberrant clone dispersal in proliferating epithelia. Dev Cell. PubMed ID: 31495693
Summary:
As epithelial tissues develop, groups of cells related by descent tend to associate in clonal populations rather than dispersing within the cell layer. While this is frequently assumed to be a result of differential adhesion, precise mechanisms controlling clonal cohesiveness remain unknown. This study employed computational simulations to modulate epithelial cell size in silico and shows that junctions between small cells frequently collapse, resulting in clone-cell dispersal among larger neighbors. Consistent with similar dynamics in vivo, it was further demonstrated that mosaic disruption of Drosophila Tor generates small cells and results in aberrant clone dispersal in developing wing disc epithelia. A geometric basis is proposed for this phenomenon, supported in part by the observation that soap-foam cells exhibit similar size-dependent junctional rearrangements. Combined, these results establish a link between cell-size pleomorphism and the control of epithelial cell packing, with potential implications for understanding tumor cell dispersal in human disease.
Sharifkhodaei, Z., Gilbert, M. M. and Auld, V. J. (2019). Scribble and Discs-large mediate tricellular junction formation. Development. PubMed ID: 31444218
Summary:
Junctional complexes that mediate cell adhesion are key to epithelial integrity, cell division and permeability barriers formation. In Drosophila the scaffolding proteins Scribble (Scrib) and Discs-large (Dlg) are key regulators of epithelial polarity, proliferation, assembly of junctions, and protein trafficking. This study found that Scrib and Dlg are necessary for the formation of the tricellular junction (TCJ), a unique junction that forms in epithelia at the convergence of three neighbouring cells. Scrib and Dlg are in close proximity with the TCJ proteins Gliotactin (Gli) and Bark-beetle (Bark) and both are required for TCJ protein recruitment. Loss of Bark or Gli lead to the basolateral spread of the TCJ complex at the cell corners. Loss of the septate junction proteins NrxIV and the Na(+)/K(+) ATPase also resulted in the basolateral spread of the entire TCJ complex at the cell corners. The Scrib PDZ1-2 domains and the Dlg GUK domain are necessary for Bark and Gli localization to the TCJ. Overall, a model is proposed where Scrib and Dlg are key components of the TCJ, and form a complex with Bark and Gli.

Thursday, October 10th

Sancer, G., Kind, E., Plazaola-Sasieta, H., Balke, J., Pham, T., Hasan, A., Munch, L. O., Courgeon, M., Mathejczyk, T. F. and Wernet, M. F. (2019). Modality-specific circuits for skylight orientation in the fly visual system. Curr Biol 29(17): 2812-2825. PubMed ID: 31402302 In the fly optic lobe, approximately 800 highly stereotypical columnar microcircuits are arranged retinotopically to process visual information. Differences in cellular composition and synaptic connectivity within functionally specialized columns remain largely unknown. This study describes the cellular and synaptic architecture in medulla columns located downstream of photoreceptors in the dorsal rim area (DRA), where linearly polarized skylight is detected for guiding orientation responses. Only in DRA medulla columns both R7 and R8 photoreceptors target to the bona fide R7 target layer where they form connections with previously uncharacterized, modality-specific Dm neurons: two morphologically distinct DRA-specific cell types (termed Dm-DRA1 and Dm-DRA2) stratify in separate sublayers and exclusively contact polarization-sensitive DRA inputs, while avoiding overlaps with color-sensitive Dm8 cells. Using the activity-dependent GRASP and trans-Tango techniques, it was confirmed that DRA R7 cells are synaptically connected to Dm-DRA1, whereas DRA R8 form synapses with Dm-DRA2. Finally, using live imaging of ingrowing pupal photoreceptor axons, it was shown that DRA R7 and R8 termini reach layer M6 sequentially, thus separating the establishment of different synaptic connectivity in time. It is proposed that a duplication of R7-->Dm circuitry in DRA ommatidia serves as an ideal adaptation for detecting linearly polarized skylight using orthogonal e-vector analyzers. Svendsen, P. C., Phillips, L. A., Deshwar, A. R., Ryu, J. R., Najand, N. and Brook, W. J. (2019). The selector genes midline and H15 control ventral leg pattern by both inhibiting Dpp signaling and specifying ventral fate. Dev Biol. PubMed ID: 31299230
Summary:
mid and H15 encode Tbx20 transcription factors that specify ventral pattern in the Drosophila leg. There are at least two pathways for mid and H15 specification of ventral fate. In the first pathway, mid and H15 negatively regulate Dpp, the dorsal signal in leg development. mid and H15 block the dorsalizing effects of Dpp signaling in the ventral leg. In loss- and gain-of-function experiments in imaginal discs, this study shows that mid and H15 block the accumulation of phospho-Mad, the activated form of the Drosophila pSmad1/5 homolog. In a second pathway, mid and H15 must also directly promote ventral fate because simultaneously blocking Dpp signaling in mid H15 mutants does not rescue the ventral to dorsal transformation in most ventral leg structures. mid and H15 act as transcriptional repressors in ventral leg development. The two genes repress the Dpp target gene Dad, the laterally expressed gene Upd, and the mid VLE enhancer. This repression depends on the eh1 domain, a binding site for the Groucho co-repressor, and is likely direct because Mid localizes to target gene enhancers in PCR-ChIP assays. A mid allele mutant for the repressing domain (eh1), mid(eh1), was found to be compromised in gain-of-function assays and in rescue of mid H15 loss-of-function. It is proposed that mid and H15 specify ventral fate through inhibition of Dpp signaling and through coordinating the repression of genes in the ventral leg.

., Sousa-Victor, P. and Jasper, H. (2019). The WT1-like transcription factor Klumpfuss maintains lineage commitment of enterocyte progenitors in the Drosophila intestine. Nat Commun 10(1): 4123. PubMed ID: 31511511
Summary:
In adult epithelial stem cell lineages, the precise differentiation of daughter cells is critical to maintain tissue homeostasis. Notch signaling controls the choice between absorptive and entero-endocrine cell differentiation in both the mammalian small intestine and the Drosophila midgut, yet how Notch promotes lineage restriction remains unclear. This study describes a role for the transcription factor Klumpfuss (Klu) in restricting the fate of enteroblasts (EBs) in the Drosophila intestine. Klu is induced in Notch-positive EBs and its activity restricts cell fate towards the enterocyte (EC) lineage. Transcriptomics and DamID profiling show that Klu suppresses enteroendocrine (EE) fate by repressing the action of the proneural gene Scute, which is essential for EE differentiation. Loss of Klu results in differentiation of EBs into EE cells. These findings provide mechanistic insight into how lineage commitment in progenitor cell differentiation can be ensured downstream of initial specification cues.

Deng, M., Wang, Y., Zhang, L., Yang, Y., Huang, S., Wang, J., Ge, H., Ishibashi, T. and Yan, Y. (2019). Single cell transcriptomic landscapes of pattern formation, proliferation and growth in Drosophila wing imaginal discs. Development. PubMed ID: 31455604
Summary:
Organ formation relies on the orchestration of pattern formation, proliferation and growth during development. How these processes are integrated at individual cell level remains unclear. Studies using Drosophila wing imaginal discs as a model system have provided valuable insights into pattern formation, growth control and regeneration in the past decades. This study provides single cell transcriptomic landscapes of pattern formation, proliferation and growth of wing imaginal discs. Patterning information is robustly maintained in the single cell transcriptomic data and can provide reference matrices to computationally map single cells into discrete spatial domains. Assignment of wing disc single cells to spatial sub-regions facilitates examination of patterning refinement processes. Single cells were clustered into different proliferation and growth states, and the correlation was evaluated between cell proliferation/growth states and spatial patterning. Furthermore, the single cell transcriptomic analysis allowed quantitative examination of the disturbance of differentiation, proliferation and growth in a well-established tumor model. A database explores these datasets at: http://drosophilayanlab-virtual-wingdisc.ust.hk:3838/v2/.
Pan, X., Neufeld, T. P. and O'Connor, M. B. (2019). A tissue- and temporal-specific autophagic switch controls Drosophila pre-metamorphic nutritional checkpoints. Curr Biol 29(17): 2840-2851.e2844. PubMed ID: 31422886
Summary:
Properly timed production of steroid hormones by endocrine tissues regulates juvenile-to-adult transitions in both mammals (puberty) and holometabolous insects (metamorphosis). Nutritional conditions influence the temporal control of the transition, but the mechanisms responsible are ill defined. This study demonstrates that autophagy acts as an endocrine organ-specific, nutritionally regulated gating mechanism to help ensure productive metamorphosis in Drosophila. Autophagy in the endocrine organ is specifically stimulated by nutrient restriction at the early, but not the late, third-instar larva stage. The timing of autophagy induction correlates with the nutritional checkpoints, which inhibit precocious metamorphosis during nutrient restriction in undersized larvae. Suppression of autophagy causes dysregulated pupariation of starved larvae, which leads to pupal lethality, whereas forced autophagy induction results in developmental delay/arrest in well-fed animals. Induction of autophagy disrupts production of the steroid hormone ecdysone at the time of pupariation not by destruction of hormone biosynthetic capacity but rather by limiting the availability of the steroid hormone precursor cholesterol in the endocrine cells via a lipophagy mechanism. Interestingly, autophagy in the endocrine organ functions by interacting with the endolysosome system, yet shows multiple features not fully consistent with a canonical autophagy process. Taken together, these findings demonstrate an autophagy mechanism in endocrine cells that helps shape the nutritional checkpoints and guarantee a successful juvenile-to-adult transition in animals confronting nutritional stress.
Rotelli, M. D., Bolling, A. M., Killion, A. W., Weinberg, A. J., Dixon, M. J. and Calvi, B. R. (2019). An RNAi screen for genes required for growth of Drosophila wing tissue. G3 (Bethesda). PubMed ID: 31387856
Summary:
Cell division and tissue growth must be coordinated with development. Defects in these processes are the basis for a number of diseases, including developmental malformations and cancer. For this study an unbiased RNAi screen was conducted for genes that are required for growth in the Drosophila wing, using GAL4-inducible short hairpin RNA (shRNA) fly strains made by the Drosophila RNAi Screening Center. shRNA expression down the center of the larval wing disc using dpp-GAL4, and the central region of the adult wing was then scored for tissue growth and wing hair morphology. Out of 4,753 shRNA crosses that survived to adulthood, 18 had impaired wing growth. FlyBase and the new Alliance of Genome Resources knowledgebases were used to determine the known or predicted functions of these genes and the association of their human orthologs with disease. The function of eight of the genes identified has not been previously defined in Drosophila. The genes identified included those with known or predicted functions in cell cycle, chromosome segregation, morphogenesis, metabolism, steroid processing, transcription, and translation. All but one of the genes are similar to those in humans, and many are associated with disease. Knockdown of lin-52, a subunit of the Myb-MuvB transcription factor, or betaNACtes6, a gene involved in protein folding and trafficking, resulted in a switch from cell proliferation to an endoreplication growth program through which wing tissue grew by an increase in cell size (hypertrophy). It is anticipated that further analysis of these genes will reveal new mechanisms that regulate tissue growth during development.

Wednesday, October 9th - Adult Neural Development

Katana, R., Guan, C., Zanini, D., Larsen, M. E., Giraldo, D., Geurten, B. R. H., Schmidt, C. F., Britt, S. G. and Gopfert, M. C. (2019). Chromophore-independent roles of opsin apoproteins in Drosophila mechanoreceptors. Curr Biol 29(17): 2961-2969. PubMed ID: 31447373
Summary:
Rhodopsins, the major light-detecting molecules of animal visual systems, consist of opsin apoproteins that covalently bind a retinal chromophore with a conserved lysine residue. In addition to capturing photons, this chromophore contributes to rhodopsin maturation, trafficking, and stabilization, and defects in chromophore synthesis and recycling can cause dysfunction of the retina and dystrophy. Indications that opsin apoproteins alone might have biological roles have come from archaebacteria and platyhelminths, which present opsin-like proteins that lack the chromophore binding site and are deemed to function independently of light. Light-independent sensory roles have been documented for Drosophila opsins, yet also these unconventional opsin functions are thought to require chromophore binding. Unconjugated opsin apoproteins act as phospholipid scramblases in mammalian photoreceptor disks, yet chromophore-independent roles of opsin apoproteins outside of eyes have not been described. Drosophila chordotonal mechanoreceptors require opsins, and this study finds that their function remains uncompromised by nutrient carotenoid depletion. Disrupting carotenoid uptake and cleavage also left the mechanoreceptors unaffected, and manipulating the chromophore attachment site of the fly's major visual opsin Rh1 impaired photoreceptor, but not mechanoreceptor, function. Notwithstanding this chromophore independence, some proteins that process and recycle the chromophore in the retina are also required in mechanoreceptors, including visual cycle components that recycle the chromophore upon its photoisomerization. These results thus establish biological function for unconjugated opsin apoproteins outside of eyes and, in addition, document chromophore-independent roles for chromophore pathway components.
Palavicino-Maggio, C. B., Chan, Y. B., McKellar, C. and Kravitz, E. A. (2019). A small number of cholinergic neurons mediate hyperaggression in female Drosophila. Proc Natl Acad Sci U S A 116(34): 17029-17038. PubMed ID: 31391301
Summary:
In the Drosophila model of aggression, males and females fight in same-sex pairings, but a wide disparity exists in the levels of aggression displayed by the 2 sexes. A screen of Drosophila Flylight Gal4 lines by driving expression of the gene coding for the temperature sensitive dTRPA1 channel, yielded a single line (GMR26E01-Gal4) displaying greatly enhanced aggression when thermoactivated. Targeted neurons were widely distributed throughout male and female nervous systems, but the enhanced aggression was seen only in females. No effects were seen on female mating behavior, general arousal, or male aggression. The enhancement was quantified by measuring fight patterns characteristic of female and male aggression and confirmed that the effect was female-specific. To reduce the numbers of neurons involved, an intersectional approach was used with a library of enhancer trap flp-recombinase lines. Several crosses reduced the populations of labeled neurons, but only 1 cross yielded a large reduction while maintaining the phenotype. Of particular interest was a small group (2 to 4 pairs) of neurons in the approximate position of the pC1 cluster important in governing male and female social behavior. Female brains have approximately 20 doublesex (dsx)-expressing neurons within pC1 clusters. Using dsx (FLP) instead of 357 (FLP) for the intersectional studies, it was found that the same 2 to 4 pairs of neurons likely were identified with both. These neurons were cholinergic and showed no immunostaining for other transmitter compounds. Blocking the activation of these neurons blocked the enhancement of aggression.
Meng, J. L., Marshall, Z. D., Lobb-Rabe, M. and Heckscher, E. S. (2019). How prolonged expression of Hb, a temporal transcription factor, re-wires locomotor circuits. Elife 8. PubMed ID: 31502540
Summary:
How circuits assemble starting from stem cells is a fundamental question in developmental neurobiology. This study tested the hypothesis that, in neuronal stem cells, temporal transcription factors predictably control neuronal terminal features and circuit assembly. Using the Drosophila motor system, expression of the classic temporal transcription factor Hunchback (Hb) was manipulated specifically in the NB7-1 stem cell, which produces U motor neurons (MNs), and then dendrite morphology and neuromuscular synaptic partnerships were monitored. Prolonged expression of Hb leads to transient specification of U MN identity, and that embryonic molecular markers do not accurately predict U MN terminal features. Nonetheless, the data show Hb acts as a potent regulator of neuromuscular wiring decisions. These data introduce important refinements to current models, show that molecular information acting early in neurogenesis as a switch to control motor circuit wiring and provide novel insight into the relationship between stem cell and circuit.
Nisha, Aggarwal, P. and Sarkar, S. (2019). Adequate expression of Globin1 is required for development and maintenance of nervous system in Drosophila. Mol Cell Neurosci 100: 103398. PubMed ID: 31472221
Summary:
This study investigates the role of globin1 (glob1) in the development and maintenance of the nervous system in Drosophila. Ubiquitous or pan-neuronal downregulation of glob1 causes partial lethality and mis-positioning of various neural-progenitor cells present in the embryonic midline cell clusters. Subsequently, profound expression of Glob1 was noted in the outer proliferation center of larval brain and photoreceptor axons of optic stalk. The overall arrangement of photoreceptor axons and stereotype positioning of neuroblast cells present in the central region of the brain were severally affected due to reduced expression of glob1. In addition, such larvae and surviving adults develop significant neuro-muscular disabilities. For the first time, this study suggests a novel role of glob1 in development and maintenance of the nervous system adding a new dimension to the functional significance of the multi-tasking glob1 gene in Drosophila.
Plazaola-Sasieta, H., Zhu, Q., Gaitan-Penas, H., Rios, M., Estevez, R. and Morey, M. (2019). Drosophila ClC-a is required in glia of the stem cell niche for proper neurogenesis and wiring of neural circuits. Glia. PubMed ID: 31479171
Summary:
Glial cells form part of the neural stem cell niche and express a wide variety of ion channels; however, the contribution of these channels to nervous system development is poorly understood. This study explored the function of the Drosophila ClC-a chloride channel, since its mammalian ortholog CLCN2 is expressed in glial cells, and defective channel function results in leukodystrophies, which in humans are accompanied by cognitive impairment. ClC-a was expressed in the niche in cortex glia, which are closely associated with neurogenic tissues. Characterization of loss-of-function ClC-a mutants revealed that these animals had smaller brains and widespread wiring defects. ClC-a is required in cortex glia for neurogenesis in neuroepithelia and neuroblasts, and defects were identified in a neuroblast lineage that generates guidepost glial cells essential for photoreceptor axon guidance. It is propose that glia-mediated ionic homeostasis could nonautonomously affect neurogenesis, and consequently, the correct assembly of neural circuits.
Liu, W., Ganguly, A., Huang, J., Wang, Y., Ni, J. D., Gurav, A. S., Aguilar, M. A. and Montell, C. (2019). Neuropeptide F regulates courtship in Drosophila through a male-specific neuronal circuit. Elife 8. PubMed ID: 31403399
Summary:
Male courtship is provoked by perception of a potential mate. In addition, the likelihood and intensity of courtship are influenced by recent mating experience, which affects sexual drive. Using Drosophila melanogaster, this study found that the homolog of mammalian neuropeptide Y, neuropeptide F (NPF), and a cluster of male-specific NPF (NPF(M)) neurons, regulate courtship through affecting courtship drive. Disrupting NPF signaling produces sexually hyperactive males, which are resistant to sexual satiation, and whose courtship is triggered by sub-optimal stimuli. This study found that NPF(M) neurons make synaptic connections with P1 neurons, which comprise the courtship decision center. Activation of P1 neurons elevates NPF(M) neuronal activity, which then act through NPF receptor neurons to suppress male courtship, and maintain the proper level of male courtship drive.

Tuesday, October 8th - Disease Models

Park, S., Park, S. K., Watanabe, N., Hashimoto, T., Iwatsubo, T., Shelkovnikova, T. A. and Liebman, S. W. (2019). Calcium-responsive transactivator (CREST) toxicity is rescued by loss of PBP1/ATXN2 function in a novel yeast proteinopathy model and in transgenic flies. PLoS Genet 15(8): e1008308. PubMed ID: 31390360
Summary:
Proteins associated with familial neurodegenerative disease often aggregate in patients' neurons. Several such proteins, e.g. TDP-43, aggregate and are toxic when expressed in yeast. Deletion of the ATXN2 ortholog, PBP1, reduces yeast TDP-43 toxicity, which led to identification of ATXN2 as an amyotrophic lateral sclerosis (ALS) risk factor and therapeutic target. Likewise, new yeast neurodegenerative disease models could facilitate identification of other risk factors and targets. Mutations in SS18L1, encoding the calcium-responsive transactivator (CREST) chromatin-remodeling protein, are associated with ALS. This study shows that CREST is toxic in yeast and forms nuclear and occasionally cytoplasmic foci that stain with Thioflavin-T, a dye indicative of amyloid-like protein. Like the yeast chromatin-remodeling factor SWI1, CREST inhibits silencing of FLO genes. Toxicity of CREST is enhanced by the [PIN+] prion and reduced by deletion of the HSP104 chaperone required for the propagation of many yeast prions. Likewise, deletion of PBP1 reduced CREST toxicity and aggregation. In accord with the yeast data, this study shows that the Drosophila ortholog of human ATXN2, dAtx2, is a potent enhancer of CREST toxicity. Downregulation of dAtx2 in flies overexpressing CREST in retinal ganglion cells was sufficient to largely rescue the severe degenerative phenotype induced by human CREST. Overexpression caused considerable co-localization of CREST and PBP1/ATXN2 in cytoplasmic foci in both yeast and mammalian cells. Thus, co-aggregation of CREST and PBP1/ATXN2 may serve as one of the mechanisms of PBP1/ATXN2-mediated toxicity. These results extend the spectrum of ALS associated proteins whose toxicity is regulated by PBP1/ATXN2, suggesting that therapies targeting ATXN2 may be effective for a wide range of neurodegenerative diseases.
Lang, S., Hilsabeck, T. A., Wilson, K. A., Sharma, A., Bose, N., Brackman, D. J., Beck, J. N., Chen, L., Watson, M. A., Killilea, D. W., Ho, S., Kahn, A., Giacomini, K., Stoller, M. L., Chi, T. and Kapahi, P. (2019). A conserved role of the insulin-like signaling pathway in diet-dependent uric acid pathologies in Drosophila melanogaster. PLoS Genet 15(8): e1008318. PubMed ID: 31415568
Summary:
Elevated uric acid (UA) is a key risk factor for many disorders, including metabolic syndrome, gout and kidney stones. Despite frequent occurrence of these disorders, the genetic pathways influencing UA metabolism and the association with disease remain poorly understood. In humans, elevated UA levels resulted from the loss of the of the urate oxidase (Uro) gene around 15 million years ago. Therefore, this study used a Drosophila melanogaster model with reduced expression of the orthologous Uro gene to study the pathogenesis arising from elevated UA. Reduced Uro expression in Drosophila resulted in elevated UA levels, accumulation of concretions in the excretory system, and shortening of lifespan when reared on diets containing high levels of yeast extract. Furthermore, high levels of dietary purines, but not protein or sugar, were sufficient to produce the same effects of shortened lifespan and concretion formation in the Drosophila model. The insulin-like signaling (ILS) pathway has been shown to respond to changes in nutrient status in several species. This study observed that genetic suppression of ILS genes reduced both UA levels and concretion load in flies fed high levels of yeast extract. Further support for the role of the ILS pathway in modulating UA metabolism stems from a human candidate gene study identifying SNPs in the ILS genes AKT2 and FOXO3 being associated with serum UA levels or gout. Additionally, inhibition of the NADPH oxidase (NOX) gene rescued the reduced lifespan and concretion phenotypes in Uro knockdown flies. Thus, components of the ILS pathway and the downstream protein NOX represent potential therapeutic targets for treating UA associated pathologies, including gout and kidney stones, as well as extending human healthspan.
Park, S. Y., Seo, J. and Chun, Y. S. (2019). Targeted downregulation of kdm4a ameliorates Tau-engendered defects in Drosophila melanogaster. J Korean Med Sci 34(33): e225. PubMed ID: 31436053
Summary:
Tauopathies, a class of neurodegenerative diseases that includes Alzheimer's disease (AD), are characterized by the deposition of neurofibrillary tangles composed of hyperphosphorylated tau protein in the human brain. As abnormal alterations in histone acetylation and methylation show a cause and effect relationship with AD, this study investigated the role of several Jumonji domain-containing histone demethylase (JHDM) genes, which have yet to be studied in AD pathology. To examine alterations of several JHDM genes in AD pathology, bioinformatics analyses were performed of JHDM gene expression profiles in brain tissue samples from deceased AD patients. Furthermore, to investigate the possible relationship between alterations in JHDM gene expression profiles and AD pathology in vivo, whether tissue-specific downregulation of JHDM Drosophila homologs (kdm) can affect tau(R406W)-induced neurotoxicity using transgenic flies containing the UAS-Gal4 binary system. The expression levels of JHDM1A, JHDM2A/2B, and JHDM3A/3B were significantly higher in postmortem brain tissue from patients with AD than from non-demented controls, whereas JHDM1B mRNA levels were downregulated in the brains of patients with AD. Using transgenic flies, it was revealed that knockdown of kdm2 (homolog to human JHDM1), kdm3 (homolog to human JHDM2), kdm4a (homolog to human JHDM3A), or kdm4b (homolog to human JHDM3B) genes in the eye ameliorated the tau(R406W)-engendered defects, resulting in less severe phenotypes. However, kdm4a knockdown in the central nervous system uniquely ameliorated tau(R406W)-induced locomotion defects by restoring heterochromatin. These results suggest that downregulation of kdm4a expression may be a potential therapeutic target in AD.
Ding, Y., Kong, D., Zhou, T., Yang, N. D., Xin, C., Xu, J., Wang, Q., Zhang, H., Wu, Q., Lu, X., Lim, K., Ma, B., Zhang, C., Li, L. and Huang, W. (2019). alpha-Arbutin protects against Parkinson's disease-associated mitochondrial dysfunction in vitro and in vivo. Neuromolecular Med. PubMed ID: 31401719
Summary:
Parkinson's disease (PD), the most common neurodegenerative movement disorder, is characterized by the progressive loss of dopaminergic neurons in substantia nigra. The underlying mechanisms of PD pathogenesis have not been fully illustrated and currently PD remains incurable. Accumulating evidences suggest that mitochondrial dysfunction plays pivotal role in the dopaminergic neuronal death. Therefore, discovery of novel and safe agent for rescuing mitochondrial dysfunction would benefit PD treatment. This study demonstrated that alpha-Arbutin (Arb), a natural polyphenol extracted from Ericaceae species, displayed significant protective effect on the rotenone (Rot)-induced mitochondrial dysfunction and apoptosis of human neuroblastoma cell (SH-SY5Y). It was further found that the neuroprotective effect of Arb was associated with ameliorating oxidative stress, stabilizing of mitochondrial membrane potential, and enhancing adenosine triphosphate production. To investigate the underlying mechanism, the AMP-activated protein kinase and autophagy pathway was checked, were found to be involved in the neuroprotection of Arb. Moreover, the protective effect of Arb was explored in Drosophila PD model, and Arb was found to rescue parkin deficiency-induced motor function disability and mitochondrial abnormality of Drosophila. Taken together, this study demonstrated that Arb got excellent neuroprotective effect on PD models both in vitro and in vivo and Arb might serve as a potent therapeutic agent for the treatment of PD.
Maitra, U., Scaglione, M. N., Chtarbanova, S. and O'Donnell, J. M. (2019). Innate immune responses to paraquat exposure in a Drosophila model of Parkinson's disease. Sci Rep 9(1): 12714. PubMed ID: 31481676
Summary:
Parkinson's disease (PD) is a progressive, neurodegenerative movement disorder characterized by the loss of dopaminergic (DA) neurons. Limited understanding of the early molecular pathways associated with the demise of DA neurons, including those of inflammatory exacerbation of neurodegeneration, is a major impediment to therapeutic development. Recent studies have implicated gene-environment interactions in PD susceptibility. This study used transcriptomic profiling in a Drosophila PD model in response to paraquat (PQ)-induced oxidative stress to identify pre-symptomatic signatures of impending neuron dysfunction. RNAseq data analysis revealed extensive regulation of innate immune response genes following PQ ingestion. PQ exposure leads to the activation of the NF-kappaB transcription factor, Relish, and the stress signaling factor JNK, encoded by the gene basket in Drosophila. Relish knockdown in the dopaminergic neurons confers PQ resistance and rescues mobility defects and DA neuron loss. Furthermore, PQ-induced toxicity is mediated through the immune deficiency signaling pathway. Surprisingly, the expression of Relish-dependent anti-microbial peptide (AMPs) genes is suppressed upon PQ exposure causing increased sensitivity to Gram-negative bacterial infection. This work provides a novel link between PQ exposure and innate immune system modulation underlying environmental toxin-induced neurodegeneration, thereby underscoring the role of the innate immune system in PD pathogenesis.
Rohde, P. D., Jensen, I. R., Sarup, P. M., Orsted, M., Demontis, D., Sorensen, P. and Kristensen, T. N. (2019). Genetic signatures of drug response variability in Drosophila melanogaster. Genetics. PubMed ID: 31455722
Summary:
Knowledge of the genetic basis underlying variation in response to environmental exposures or treatment is important in many research areas. For example, knowing the set of causal genetic variants for drug response could revolutionize personalized medicine. This study used Drosophila melanogaster to investigate the genetic signature underlying behavioral variability in response to methylphenidate (MPH), a drug used in the treatment of attention-deficit/hyperactivity disorder (ADHD). A wild type D. melanogaster population was exposed to MPH and a control treatment, and an increase was observed in locomotor activity in MPH-exposed individuals. Whole-genome transcriptomic analyses revealed that the behavioral response to MPH was associated with abundant gene expression alterations. To confirm these patterns in a different genetic background, and to further advance knowledge on the genetic signature of drug response variability, a system of inbred lines, the Drosophila Genetic Reference Panel (DGRP) was used. Based on the DGRP, this study showed that the behavioral response to MPH was strongly genotype-dependent. Using an integrative genomic approach, known gene interactions were incorporated into the genomic analyses of the DGRP, and putative candidate genes for variability in drug response were identified. 71% of the investigated candidate genes were successfully analyzed by gene expression knockdown. Furthermore, it was shown that MPH has cross-generational behavioral and transcriptomic effects. These findings establish a foundation for understanding the genetic mechanisms driving genotype-specific responses to medical treatment, and highlight the opportunities that integrative genomic approaches have in optimizing medical treatment of complex diseases.

Monday October 7th - RNA and Transposons

Kneuss, E., Munafo, M., Eastwood, E. L., Deumer, U. S., Preall, J. B., Hannon, G. J. and Czech, B. (2019). Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export. Genes Dev 33(17-18): 1208-1220. PubMed ID: 31416967
Summary:
The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. This study reports that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. These data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.
Cappucci, U., Noro, F., Casale, A. M., Fanti, L., Berloco, M., Alagia, A. A., Grassi, L., Le Pera, L., Piacentini, L. and Pimpinelli, S. (2019). The Hsp70 chaperone is a major player in stress-induced transposable element activation. Proc Natl Acad Sci U S A 116(36): 17943-17950. PubMed ID: 31399546
Summary:
Previous studies have shown that heat shock stress may activate transposable elements (TEs) in Drosophila and other organisms. Such an effect depends on the disruption of a chaperone complex that is normally involved in biogenesis of Piwi-interacting RNAs (piRNAs), the largest class of germline-enriched small noncoding RNAs implicated in the epigenetic silencing of TEs. However, a satisfying picture of how chaperones could be involved in repressing TEs in germ cells is still unknown. This study shows that, in Drosophila, heat shock stress increases the expression of TEs at a posttranscriptional level by affecting piRNA biogenesis through the action of the inducible chaperone Hsp70. Stress-induced TE activation is triggered by an interaction of Hsp70 with the Hsc70-Hsp90 complex and other factors all involved in piRNA biogenesis in both ovaries and testes. Such interaction induces a displacement of all such factors to the lysosomes, resulting in a functional collapse of piRNA biogenesis. This mechanism has clear evolutionary implications. In the presence of drastic environmental changes, Hsp70 plays a key dual role in increasing both the survival probability of individuals and the genetic variability in their germ cells. The consequent increase of genetic variation in a population potentiates evolutionary plasticity and evolvability.
ElMaghraby, M. F., Andersen, P. R., Puhringer, F., Hohmann, U., Meixner, K., Lendl, T., Tirian, L. and Brennecke, J. (2019). A heterochromatin-specific RNA export pathway facilitates piRNA production. Cell 178(4): 964-979. PubMed ID: 31398345
Summary:
PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, this study uncovered an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.
Nian, X., Chen, W., Bai, W., Zhao, Z. and Zhang, Y. (2019). miR-263b controls circadian behavior and the structural plasticity of pacemaker neurons by regulating the LIM-only protein Beadex. Cells 8(8). PubMed ID: 31426557
Summary:
Circadian clocks drive rhythmic physiology and behavior to allow adaption to daily environmental changes. In Drosophila, the small ventral lateral neurons (sLNvs) are primary pacemakers that control circadian rhythms. Circadian changes are observed in the dorsal axonal projections of the sLNvs, but their physiological importance and the underlying mechanism are unclear. This study identified miR-263b as an important regulator of circadian rhythms and structural plasticity of sLNvs in Drosophila. Depletion of miR-263b (miR-263b(KO)) in flies dramatically impaired locomotor rhythms under constant darkness. Indeed, miR-263b is required for the structural plasticity of sLNvs. miR-263b regulates circadian rhythms through inhibition of expression of the LIM-only protein Beadex (Bx). Consistently, overexpression of Bx or loss-of-function mutation (BxhdpR26) phenocopied miR-263b(KO) and miR-263b overexpression in behavior and molecular characteristics. In addition, mutating the miR-263b binding sites in the Bx 3' UTR using CRISPR/Cas9 recapitulated the circadian phenotypes of miR-263b(KO) flies. Together, these results establish miR-263b as an important regulator of circadian locomotor behavior and structural plasticity.
Obrdlik, A., Lin, G., Haberman, N., Ule, J. and Ephrussi, A. (2019). The transcriptome-wide landscape and modalities of EJC binding in adult Drosophila. Cell Rep 28(5): 1219-1236.e1211. PubMed ID: 31365866
Summary:
Exon junction complex (EJC) assembles after splicing at specific positions upstream of exon-exon junctions in mRNAs of all higher eukaryotes, affecting major regulatory events. In mammalian cell cytoplasm, EJC is essential for efficient RNA surveillance, while in Drosophila, EJC is essential for localization of oskar mRNA. This study has developed a method for isolation of protein complexes and associated RNA targets (ipaRt) to explore the EJC RNA-binding landscape in a transcriptome-wide manner in adult Drosophila. The EJC was found at canonical positions, preferably on mRNAs from genes comprising multiple splice sites and long introns. Moreover, EJC occupancy is highest at junctions adjacent to strong splice sites, CG-rich hexamers, and RNA structures. Highly occupied mRNAs tend to be maternally localized and derive from genes involved in differentiation or development. These modalities, which have not been reported in mammals, specify EJC assembly on a biologically coherent set of transcripts in Drosophila.
Deryusheva, S. and Gall, J. G. (2019). Small, smaller, smallest: Minimal structural requirements for a fully functional box C/D modification guide RNA. Biomolecules 9(9). PubMed ID: 31500270
Summary:
Site-specific 2'-O-ribose methylation is an abundant post-transcriptional modification mediated by small non-coding nuclear RNAs known as box C/D modification guide RNAs. The minimal structural requirements for these guide RNAs to function in higher eukaryotes are still unclear. To address this question, a series of mutant variants of Drosophila box C/D scaRNA:MeU2-C28 was generated, and their modification guide activities were tested in the Xenopus oocyte system. The data suggest that box C/D guide RNA function requires either a terminal or an internal consensus kink-turn structure. The minimal functional box C/D guide RNA was identified. It consists of a single-domain molecule with (1) a terminal stem with a consensus kink-turn domain, (2) one box C and box D connected by a 14-nucleotide antisense element and (3) a one-nucleotide spacer between the box C and the antisense element. In this single domain RNA, the sequence of the spacer is more important than its length. It is suggested that the secondary structure of box C/D RNAs, essential for guide RNA function, is more complex than generally supposed. At the same time, the expression of functional extremely short single-domain box C/D RNAs is possible in higher eukaryotes.

Friday, October 4th - Signaling

Nil, Z., Millan, R. H., Gerbich, T., Leal, P., Yu, Z., Saraf, A., Sardiu, M., Lange, J. J., Yi, K., Unruh, J., Slaughter, B. and Si, K. (2019). Amyloid-like assembly activates a phosphatase in the developing Drosophila embryo. Cell 178(6): 1403-1420. PubMed ID: 31491385
Summary:
Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. This study reports the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-beta/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog, a protein-serine/threonine phosphatase, is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.
Li, M., Sun, S., Priest, J., Bi, X. and Fan, Y. (2019). Characterization of TNF-induced cell death in Drosophila reveals caspase- and JNK-dependent necrosis and its role in tumor suppression. Cell Death Dis 10(8): 613. PubMed ID: 31409797
Summary:
This study examined the molecular mechanisms that distinguish the forms of cell death induced by Eiger (Egr), the sole homolog of TNF in Drosophila. Expression of Egr in the developing Drosophila eye simultaneously induces apoptosis and apoptosis-independent developmental defects indicated by cellular disorganization, both of which rely on the c-Jun N-terminal kinase (JNK) signaling activity. Intriguingly, when effector caspases DrICE and Dcp-1 are defective or inhibited, expression of Egr triggers necrosis which is characterized by loss of cell membrane integrity, translucent cytoplasm, and aggregation of cellular organelles. Moreover, such Egr-induced necrosis depends on the catalytic activity of the initiator caspase Dronc and the input from JNK signaling but is independent of their roles in apoptosis. Further mosaic analysis with mutants of scribble (scrib), an evolutionarily conserved tumor suppressor gene regulating cell polarity, suggests that Egr/JNK-mediated apoptosis and necrosis establish a two-layered defense system to inhibit the oncogenic growth of scrib mutant cells. Together, this study has identified caspase- and JNK-dependent mechanisms underlying Egr-induced apoptosis versus necrosis and their fail-safe roles in tumor suppression in an intact organism in vivo.
Mascolo, E., Amoroso, N., Saggio, I., Merigliano, C. and Verni, F. (2019). Pyridoxine/pyridoxamine 5'-phosphate oxidase (Sgll/PNPO) is important for DNA integrity and glucose homeostasis maintenance in Drosophila. J Cell Physiol. PubMed ID: 31506944
Summary:
Pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PDXK) cooperate to produce pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. PDXK phosphorylates pyridoxine, pyridoxamine, and pyridoxal by producing PNP, PMP, and PLP, whereas PNPO oxidizes PNP, PMP, into PLP. Previous work has demonstrated that PDXK depletion in Drosophila and human cells impacts on glucose metabolism and DNA integrity. This study characterized sgll, the Drosophila ortholog of PNPO gene, showing that its silencing by RNA interference elicits chromosome aberrations (CABs) in brains and induces diabetic hallmarks such as hyperglycemia and small body size. In sgll(RNAi) neuroblasts CABs are largely produced by the genotoxic effect of the advanced glycation end products triggered by high glucose. As in sgll(RNAi) cells, part of PLP is still produced by PDXK activity, these data suggest that PLP dosage need to be tightly regulated to guarantee glucose homeostasis and DNA integrity.
Lee, C. W., Kwon, Y. C., Lee, Y., Park, M. Y. and Choe, K. M. (2019). cdc37 is essential for JNK pathway activation and wound closure in Drosophila. Mol Biol Cell: mbcE18120822. PubMed ID: 31483695
Summary:
Wound closure in the Drosophila larval epidermis mainly involves non-proliferative, endocyling epithelial cells. Consequently, it is largely mediated by cell growth and migration. Both cell growth and migration in Drosophila require the co-chaperone-encoding gene cdc37. Larvae lacking cdc37 in the epidermis failed to close wounds, and the cells of the epidermis failed to change cell shape and polarize. Likewise, wound-induced cell growth was significantly reduced, and correlated with a reduction in the size of the cell nucleus. The c-Jun N-terminal kinase (JNK) pathway, which is essential for wound closure, was not typically activated in injured cdc37 knockdown larvae. In addition, JNK, Hep, Mkk4, and Tak1 protein levels were reduced, consistent with previous reports showing that Cdc37 is important for the stability of various client kinases. Protein levels of the integrin beta subunit and its wound-induced protein expression were also reduced, reflecting the disruption of JNK activation, which is crucial for expression of integrin beta during wound closure. These results are consistent with a role of Cdc37 in maintaining the stability of the JNK pathway kinases, thus mediating cell growth and migration during Drosophila wound healing.
Mishra-Gorur, K., Li, D., Ma, X., Yarman, Y., Xue, L. and Xu, T. (2019). Spz/Toll-6 signal guides organotropic metastasis in Drosophila. Dis Model Mech. PubMed ID: 31477571
Summary:
Targeted cell migration plays important roles in developmental biology and disease processes including metastasis. Drosophila tumors exhibit traits characteristic of human cancers, providing a powerful model to study developmental and cancer biology. This study finds that cells derived from Drosophila eye disc tumors also display organ specific metastasis to invade receptive organs but not wing disc. Toll receptors are known to affect innate immunity and tumor inflammatory microenvironment by modulating the NF-kappaB pathway. RNAi screen and genetic analyses show that Toll-6 is required for migration and invasion of the tumor cells. Further, receptive organs express Toll-ligands, Spz family molecules, and ectopic Spz expression renders wing disc receptive to metastasis. Finally, Toll-6 promotes metastasis by activating JNK signaling, a key regulator of cell migration. Hence, this study reports Toll-6 and Spatzle as a new pair of guidance molecules mediating organ-specific metastasis behavior and highlight a novel signaling mechanism for Toll family receptors.
Katzemich, A., Long, J. Y., Panneton, V., Fisher, L., Hipfner, D. and Schock, F. (2019). Slik phosphorylation of talin T152 is crucial for proper talin recruitment and maintenance of muscle attachment in Drosophila. Development. PubMed ID: 31511253
Summary:
Talin is the major scaffold protein linking integrin receptors with the actin cytoskeleton. In Drosophila, extended talin generates a stable link between the sarcomeric cytoskeleton and the tendon matrix at muscle attachment sites. This study identified phosphorylation sites on Drosophila talin by mass spectrometry. Talin is phosphorylated in late embryogenesis when muscles differentiate, especially on T152 in the exposed loop of the F1 domain of the talin head. Localization of talin-T150/T152A is reduced at muscle attachment sites and can only partially rescue muscle attachment compared to wild type talin. Slik was identified as the kinase phosphorylating talin at T152. Slik localizes to muscle attachment sites, and the absence of Slik reduces the localization of talin at muscle attachment sites causing phenotypes similar to talin-T150/T152A. Thus, these results demonstrate that talin phosphorylation by Slik plays an important role in fine-tuning talin recruitment to integrin adhesion sites and maintaining muscle attachment.

Thursday - Chromatin

de Mello, L. R., Hamley, I. W., Castelletto, V., Garcia, B. B. M., Han, S. W., de Oliveira, C. L. P. and da Silva, E. R. (2019). Nanoscopic structure of complexes formed between DNA and the cell-penetrating peptide Penetratin. J Phys Chem B. PubMed ID: 31465229
Summary:
One of the most remarkable examples of cell-penetrating peptides (CPPs) is Penetratin, a 16-mer fragment derived from the Drosophila Antennapedia homeobox. Understanding the structure of Penetratin/DNA complexes is a key factor for the successful design of new vectors for gene delivery and may assist in optimizing molecular carriers based on CPPs. This srudy presents a comprehensive study on the nanoscale structure of noncovalent complexes formed between Penetratin and DNA. The strong cationic nature of the peptide makes it a very efficient agent for condensing DNA strands via electrostatic attraction, and this study shows that DNA condensation is accompanied by random-to-beta-sheet transitions of Penetratin secondary structure, demonstrating that nucleic acids behave as a structuring agent upon complexation. Nanoscale-resolved spectroscopy was used to provide single-particle infrared data from DNA carriers based on CPPs, and they show that the structures are stabilized by Penetratin beta-sheet cores, whereas larger DNA fractions are preferentially located in the periphery of aggregates. In-solution infrared assays indicate that phosphate diester groups are strongly affected upon DNA condensation, presumably as a consequence of charge delocalization induced by the proximity of cationic amide groups in Penetratin. The morphology is characterized by nanoassemblies with surface fractal features, and short-range order is found in the inner structure of the scaffolds. Interestingly, the formation of beads-on-a-string arrays is found, producing nanoscale architectures that resemble structures observed in early steps of chromatin condensation. A complexation pathway where DNA condensation and peptide pairing into beta-sheets are key steps for organization is proposed.
Ma, Y., McKay, D. J. and Buttitta, L. (2019). Changes in chromatin accessibility ensure robust cell cycle exit in terminally differentiated cells. PLoS Biol 17(9): e3000378. PubMed ID: 31479438
Summary:
During terminal differentiation, most cells exit the cell cycle and enter into a prolonged or permanent G0 in which they are refractory to mitogenic signals. Entry into G0 is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM). However, when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. This study provides evidence that developmentally programmed, temporal changes in chromatin accessibility at a small subset of critical cell cycle genes act to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. During terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators Cyclin E (cycE), E2F transcription factor 1 (e2f1), and string (stg). This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle reactivation, and the regions that close contain known binding sites for effectors of mitogenic signaling pathways such as Yorkie and Notch. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains unaffected, and the closing of distal enhancers at cycE, e2f1, and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. These results uncover a mechanism that acts as a cell cycle-independent timer to limit the response to mitogenic signaling and aberrant cycling in terminally differentiating tissues. In addition, a new molecular description is provided of the cross talk between cell cycle exit and terminal differentiation during metamorphosis.
Tikhonova, E., Fedotova, A., Bonchuk, A., Mogila, V., Larschan, E. N., Georgiev, P. and Maksimenko, O. (2019). The simultaneous interaction of MSL2 with CLAMP and DNA provides redundancy in the initiation of dosage compensation in Drosophila males. Development. PubMed ID: 31320325
Summary:
The binding of the Drosophila male-specific lethal dosage compensation complex (DCC) exclusively to male X chromosome provides an excellent model system to understand mechanisms of selective recruitment of protein complexes to chromatin. Previous studies showed that the male-specific organizer of the complex, MSL2, and ubiquitous DNA-binding protein CLAMP are key players in the specificity of X chromosome binding. The CXC domain of MSL2 binds to genomic sites of DCC recruitment in vitro. Another conserved domain of MSL2, named Clamp-binding domain (CBD) directly interacts with the N-terminal zinc finger domain of CLAMP. This study found that inactivation of CBD or CXC individually only modestly affected recruitment of the DCC to the X chromosome in males. However, combination of these two genetic lesions within the same MSL2 mutant resulted in an increased loss of DCC recruitment to the X chromosome. Thus, proper MSL2 positioning requires an interaction with either CLAMP or DNA to initiate dosage compensation in Drosophila males.
Gutierrez-Perez, I., Rowley, M. J., Lyu, X., Valadez-Graham, V., Vallejo, D. M., Ballesta-Illan, E., Lopez-Atalaya, J. P., Kremsky, I., Caparros, E., Corces, V. G. and Dominguez, M. (2019). Ecdysone-induced 3D chromatin reorganization involves active enhancers bound by Pipsqueak and Polycomb. Cell Rep 28(10): 2715-2727. PubMed ID: 31484080
Summary:
Evidence suggests that Polycomb (Pc) is present at chromatin loop anchors in Drosophila. Pc is recruited to DNA through interactions with the GAGA binding factors GAF and Pipsqueak (Psq). Using HiChIP in Drosophila cells, this study found that the psq gene, which has diverse roles in development and tumorigenesis, encodes distinct isoforms with unanticipated roles in genome 3D architecture. The BR-C, ttk, and bab domain (BTB)-containing Psq isoform (Psq(L)) colocalizes genome-wide with known architectural proteins. Conversely, Psq lacking the BTB domain (Psq(S)) is consistently found at Pc loop anchors and at active enhancers, including those that respond to the hormone ecdysone. After stimulation by this hormone, chromatin 3D organization is altered to connect promoters and ecdysone-responsive enhancers bound by Psq(S). These findings link Psq variants lacking the BTB domain to Pc-bound active enhancers, thus shedding light into their molecular function in chromatin changes underlying the response to hormone stimulus.
Prayitno, K., Schauer, T., Regnard, C. and Becker, P. B. (2019). Progressive dosage compensation during Drosophila embryogenesis is reflected by gene arrangement. EMBO Rep: e48138. PubMed ID: 31286660
Summary:
In Drosophila melanogaster males, X-chromosome monosomy is compensated by chromosome-wide transcription activation. This study found that complete dosage compensation during embryogenesis takes surprisingly long and is incomplete even after 10 h of development. Although the activating dosage compensation complex (DCC) associates with the X-chromosome and MOF acetylates histone H4 early, many genes are not compensated. Acetylation levels on gene bodies continue to increase for several hours after gastrulation in parallel with progressive compensation. Constitutive genes are compensated earlier than developmental genes. Remarkably, later compensation correlates with longer distances to DCC binding sites. This time-space relationship suggests that DCC action on target genes requires maturation of the active chromosome compartment.
Wooten, M., Snedeker, J., Nizami, Z. F., Yang, X., Ranjan, R., Urban, E., Kim, J. M., Gall, J., Xiao, J. and Chen, X. (2019). Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement. Nat Struct Mol Biol. PubMed ID: 31358945
Summary:
Many stem cells undergo asymmetric division to produce a self-renewing stem cell and a differentiating daughter cell. This study shows that, similarly to H3, histone H4 is inherited asymmetrically in Drosophila melanogaster male germline stem cells undergoing asymmetric division. In contrast, both H2A and H2B are inherited symmetrically. By combining super-resolution microscopy and chromatin fiber analyses with proximity ligation assays on intact nuclei, old H3 was found to be preferentially incorporated by the leading strand, whereas newly synthesized H3 is enriched on the lagging strand. Using a sequential nucleoside analog incorporation assay, a high incidence of unidirectional replication fork movement is detected in testes-derived chromatin and DNA fibers. Biased fork movement coupled with a strand preference in histone incorporation would explain how asymmetric old and new H3 and H4 are established during replication. These results suggest a role for DNA replication in patterning epigenetic information in asymmetrically dividing cells in multicellular organisms.

Wednesday, October 2nd - Embryonic Development

Dutta, S., Djabrayan, N. J., Torquato, S., Shvartsman, S. Y. and Krajnc, M. (2019). Self-similar dynamics of nuclear packing in the early Drosophila embryo. Biophys J 117(4): 743-750. PubMed ID: 31378311
Summary:
Embryonic development starts with cleavages, a rapid sequence of reductive divisions that result in an exponential increase of cell number without changing the overall size of the embryo. In Drosophila, the final four rounds of cleavages occur at the surface of the embryo and give rise to approximately 6000 nuclei under a common plasma membrane. This study used live imaging to study the dynamics of this process and to characterize the emergent nuclear packing in this system. The characteristic length scale of the internuclear interaction was shown to scale with the density, which allows the densifying embryo to sustain the level of structural order at progressively smaller length scales. This is different from nonliving materials, which typically undergo disorder-order transition upon compression. To explain this dynamics, a particle-based model was used that accounts for density-dependent nuclear interactions and synchronous divisions. The pair statistics of the disordered packings observed in embryos was reproduced, and the scaling relation between the characteristic length scale and the density both in real and reciprocal space was recovered. This result reveals how the embryo can robustly preserve the nuclear-packing structure while being densified. In addition to providing quantitative description of self-similar dynamics of nuclear packings, this model generates dynamic meshes for the computational analysis of pattern formation and tissue morphogenesis.
Kondo, T. and Hayashi, S. (2019). Two-step regulation of trachealess ensures tight coupling of cell fate with morphogenesis in the Drosophila trachea. Elife 8. PubMed ID: 31439126
Summary:
During organogenesis, inductive signals cause cell differentiation and morphogenesis. However, how these phenomena are coordinated to form functional organs is poorly understood. This study shows that cell differentiation of the Drosophila trachea is sequentially determined in two steps and that the second step is synchronous with the invagination of the epithelial sheet. The master gene trachealess is dispensable for the initiation of invagination, while it is essential for maintaining the invaginated structure, suggesting that tracheal morphogenesis and differentiation are separately induced. trachealess expression starts in bipotential tracheal/epidermal placode cells. After invagination, its expression is maintained in the invaginated cells but is extinguished in the remaining sheet cells. A trachealess cis-regulatory module that shows both tracheal enhancer activity and silencer activity in the surface epidermal sheet was identified. It is proposed that the coupling of trachealess expression with the invaginated structure ensures that only invaginated cells canalize robustly into the tracheal fate.
Gracia, M., Theis, S., Proag, A., Gay, G., Benassayag, C. and Suzanne, M. (2019). Mechanical impact of epithelial-mesenchymal transition on epithelial morphogenesis in Drosophila. Nat Commun 10(1): 2951. PubMed ID: 31273212
Summary:
Epithelial-mesenchymal transition (EMT) is an essential process both in physiological and pathological contexts. Intriguingly, EMT is often associated with tissue invagination during development; however, the impact of EMT on tissue remodeling remain unexplored. This study shows that at the initiation of the EMT process, cells produce an apico-basal force, orthogonal to the surface of the epithelium, that constitutes an important driving force for tissue invagination in Drosophila. When EMT is ectopically induced, cells starting their delamination generate an orthogonal force and induce ectopic folding. Similarly, during mesoderm invagination, cells undergoing EMT generate an apico-basal force through the formation of apico-basal structures of myosin II. Using both laser microdissection and in silico physical modelling, this study shows that mesoderm invagination does not proceed if apico-basal forces are impaired, indicating that they constitute driving forces in the folding process. Altogether, these data reveal the mechanical impact of EMT on morphogenesis.
Bailles, A., Collinet, C., Philippe, J. M., Lenne, P. F., Munro, E. and Lecuit, T. (2019). Genetic induction and mechanochemical propagation of a morphogenetic wave. Nature 572(7770): 467-473. PubMed ID: 31413363
Summary:
Tissue morphogenesis arises from coordinated changes in cell shape driven by actomyosin contractions. Patterns of gene expression regionalize cell behaviours by controlling actomyosin contractility. This study reports two modes of control over Rho1 and myosin II (MyoII) activation in the Drosophila endoderm. First, Rho1-MyoII are induced in a spatially restricted primordium via localized transcription of the G-protein-coupled receptor ligand Fog. Second, a tissue-scale wave of Rho1-MyoII activation and cell invagination progresses anteriorly away from the primordium. The wave does not require sustained gene transcription, and is not governed by regulated Fog delivery. Instead, MyoII inhibition blocks Rho1 activation and propagation, revealing a mechanical feedback driven by MyoII. MyoII activation and invagination in each row of cells drives adhesion to the vitelline membrane mediated by integrins, apical spreading, MyoII activation and invagination in the next row. Endoderm morphogenesis thus emerges from local transcriptional initiation and a mechanically driven cycle of cell deformation.
George, J. and Jacobs, H. T. (2019). Germline knockdown of spargel (PGC-1) produces embryonic lethality in Drosophila. Mitochondrion. PubMed ID: 31473309
Summary:
The PGC-1 transcriptional coactivators have been proposed as master regulators of mitochondrial biogenesis and energy metabolism. This study shows that the single member of the family in Drosophila, spargel (srl) has an essential role in early development. Female germline-specific RNAi knockdown resulted in embryonic semilethality. Embryos were small, with most suffering a catastrophic derangement of cellularization and gastrulation, although genes dependent on localized determinants were expressed normally. The abundance of mtDNA, representative mitochondrial proteins and mRNAs were not decreased in knockdown ovaries or embryos, indicating that srl has a more general role in early development than specifically promoting mitochondrial biogenesis.
Skouloudaki, K., Christodoulou, I., Khalili, D., Tsarouhas, V., Samakovlis, C., Tomancak, P., Knust, E. and Papadopoulos, D. K. (2019). Yorkie controls tube length and apical barrier integrity during airway development. J Cell Biol. PubMed ID: 31315941
Summary:
Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. This study shows that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the delta-aminolevulinate synthase gene (Alas). It is concluded that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality.

Tuesday, October 1st - Oogenesis and Spermatogenesis

Hu, Q. and Wolfner, M. F. (2019). The Drosophila Trpm channel mediates calcium influx during egg activation. Proc Natl Acad Sci U S A. PubMed ID: 31427540
Summary:
Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. This study combined inhibitor tests, germ-line-specific RNAi knockdown, and germ-line-specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. A reduction was observed in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, these results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation.
Hopkins, B. R., et al. (2019). Divergent allocation of sperm and the seminal proteome along a competition gradient in Drosophila melanogaster. Proc Natl Acad Sci U S A 116(36): 17925-17933. PubMed ID: 31431535
Summary:
Sperm competition favors large, costly ejaculates, and theory predicts the evolution of allocation strategies that enable males to plastically tailor ejaculate expenditure to sperm competition threat. While greater sperm transfer in response to a perceived increase in the risk of sperm competition is well-supported, there is only a poor understanding of whether males (i) respond to changes in perceived intensity of sperm competition, (ii) use the same allocation rules for sperm and seminal fluid, and (iii) experience changes in current and future reproductive performance as a result of ejaculate compositional changes. Combining quantitative proteomics with fluorescent sperm labeling, this study shows that Drosophila melanogaster males exercise independent control over the transfer of sperm and seminal fluid proteins (SFPs) under different levels of male-male competition. While sperm transfer peaks at low competition, consistent with some theoretical predictions based on sperm competition intensity, the abundance of transferred SFPs generally increases at high competition levels. However, it was found that clusters of SFPs vary in the directionality and sensitivity of their response to competition, promoting compositional change in seminal fluid. By tracking the degree of decline in male mating probability and offspring production across successive matings, evidence is provided that ejaculate compositional change represents an adaptive response to current sperm competition, but one that comes at a cost to future mating performance. This work reveals a previously unknown divergence in ejaculate component allocation rules, exposes downstream costs of elevated ejaculate investment, and ultimately suggests a central role for ejaculate compositional plasticity in sexual selection.
Knapp, E. M., Li, W. and Sun, J. (2019). Downregulation of homeodomain protein Cut is essential for follicle maturation and ovulation. Development. PubMed ID: 31444217
Summary:
Proper development and maturation of a follicle is essential for successful ovulation and reproduction; however, molecular mechanisms for follicle maturation, particularly for somatic follicle cell differentiation, are poorly understood. During Drosophila oogenesis, the somatic follicle cells encasing oocytes undergo two distinct well-established transitions: the mitotic to endocycle switch at stage 6/7 and the endocycle to gene amplification switch at stage10A/10B. This study identified a novel third follicle cell transition that occurs in the final stages of oogenesis (stage 13/14). This late follicle cell transition is characterized by a downregulation of the homeodomain transcription factor Cut and the zinc-finger transcription factor Tramtrack-69 (Ttk69), and an upregulation of the transcription factor Hindsight (Hnt). Inducing expression of Cut in stage 14 follicle cells is sufficient to inhibit follicle rupture and ovulation through its negative regulation of Hnt and promotion of Ttk69 expression. This work illustrates the importance of the stage13/14 transition for follicle maturation and demonstrates the complex regulation required for somatic follicle cells to differentiate into a state primed for follicle rupture and ovulation.
Biwot, J. C., Zhang, H. B., Chen, M. Y. and Wang, Y. F. (2019). A new function of immunity-related gene Zn72D in male fertility of Drosophila melanogaster. Arch Insect Biochem Physiol: e21612. PubMed ID: 31482645
Summary:
The Drosophila zinc finger protein Zn72D was first identified as being involved in phagocytosis, indicating to have a role in immunity. It was then demonstrated to have a function in RNA splicing and dosage compensation. This study discovered a new function of Zn72D in male fertility. Knockdown of Zn72D in fly testes caused an extremely low egg hatch rate. Immunofluorescence staining of Zn72D knockdown testes exhibited scattered spermatid nuclei and no actin cones or individualization complexes (ICs) during spermiogenesis, whereas the early-stage germ cells and the spermatocytes were observed clearly. There were no mature sperms in the seminal vesicles of Zn72D knockdown fly testes, although a few sperms could be found close to the seminal vesicle. It was further shown that many cytoskeleton-related genes were significantly downregulated in fly testes due to Zn72D knockdown. Taken together these findings suggest that Zn72D may have an important function in spermatogenesis by sustaining the cytoskeleton-based morphogenesis and individualization thus ensuring the proper formation of sperm in D. melanogaster.
Durdevic, Z. and Ephrussi, A. (2019). Germ cell lineage homeostasis in Drosophila requires the Vasa RNA helicase. Genetics. PubMed ID: 31484689
Summary:
The conserved RNA helicase Vasa is required for germ cell development in many organisms. In Drosophila melanogaster loss of piRNA pathway components, including Vasa, causes Chk2-dependent oogenesis arrest. However, whether the arrest is due to Chk2-signaling at a specific stage, and whether continuous Chk2-signaling is required for the arrest was unknown. This study shows that absence of Vasa during the germarial stages causes Chk2-dependent oogenesis arrest. Additionally, the age-dependent decline of the ovariole number is reported, both in flies lacking Vasa expression only in the germarium and in loss-of-function vasa mutant flies. Chk2 activation exclusively in the germarium is sufficient to interrupt oogenesis and to reduce ovariole number in aging flies. Once induced in the germarium, Chk2-mediated arrest of germ cell development cannot be overcome by restoration of Vasa or by down-regulation of Chk2 in the arrested egg-chambers. These findings, together with the identity of Vasa-associated proteins identified in this study, demonstrate an essential role of the helicase in the germ cell lineage maintenance and indicate a function of Vasa in germline stem cell homeostasis.
Jo, K. H., Jaiswal, A., Khanal, S., Fishman, E. L., Curry, A. N. and Avidor-Reiss, T. (2019). Poc1B and Sas-6 function together during the atypical centriole formation in Drosophila melanogaster. Cells 8(8). PubMed ID: 31387336
Summary:
Insects and mammals have atypical centrioles in their sperm. However, it is unclear how these atypical centrioles form. Drosophila melanogaster sperm has one typical centriole called the giant centriole (GC) and one atypical centriole called the proximal centriole-like structure (PCL). During early sperm development, centriole duplication factors such as Ana2 and Sas-6 are recruited to the GC base to initiate PCL formation. The centriolar protein, Poc1B, is also recruited at this initiation stage, but its precise role during PCL formation is unclear. This study shows that Poc1B recruitment was dependent on Sas-6, that Poc1B had effects on cellular and PCL Sas-6, and that Poc1B and Sas-6 were colocalized in the PCL/centriole core. These findings suggest that Sas-6 and Poc1B interact during PCL formation. Co-overexpression of Ana2 and Sas-6 induced the formation of ectopic particles that contained endogenous Poc1 proteins and were composed of PCL-like structures. These structures were disrupted in Poc1 mutant flies, suggesting that Poc1 proteins stabilize the PCL-like structures. Lastly, Poc1B and Sas-6 co-overexpression also induced the formation of PCL-like structures, suggesting that they can function together during the formation of the PCL. Overall, these findings suggest that Poc1B and Sas-6 function together during PCL formation.
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