Tissue, cell type and stage-specific ectopic gene expression and RNAi induction in the Drosophila testis

The Drosophila testis has numerous advantages for the study of basic cellular processes, as production of sperm requires a highly orchestrated and complex combination of morphological changes and developmentally regulated transitions. Experimental genetics using Drosophila melanogaster has advanced dramatically with the advent of systems for ectopic expression of genetic elements in specific cells. However the genetic tools used in Drosophila research have rarely been generated with the testes in mind, and the utility of relatively few systems has been documented for this tissue. Here I will summarize ectopic expression systems that are known to work for the testis, and provide advice for selection of the most appropriate expression system in specific experimental situations

The Wnt receptor Ryk is a negative regulator of mammalian dendrite morphogenesis

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia (Grants 1061512, 1063080). ML and KS were supported by an Australian Postgraduate Award or a University of Queensland International Scholarship, respectively. Imaging work was performed in the Queensland Brain Institute’s Advanced Microscopy Facility and generously supported by an ARC LIEF grant (LE130100078). We thank Assoc. Prof. Julian Heng (Harry Perkins Institute of Medical Research, Perth, Australia) for providing the pCA-ß-EGFPm5-Silencer 3 vector, Prof. Joseph LoTurco (University of Connecticut, USA) for the piggyBAC vector, and Prof. Steven Stacker (Peter MacCallum Cancer Centre, Melbourne, Australia) for providing the Ryk knockout mice and the full-length Ryk plasmid. We are also grateful to Mr Luke Hammond for expert advice on microscopy and Ms Rowan Tweedale for critical reading of the manuscript.Peer reviewedPublisher PD

Mutations in Drosophila Greatwall/Scant Reveal Its Roles in Mitosis and Meiosis and Interdependence with Polo Kinase

Polo is a conserved kinase that coordinates many events of mitosis and meiosis, but how it is regulated remains unclear. Drosophila females having only one wild-type allele of the polo kinase gene and the dominant Scant mutation produce embryos in which one of the centrosomes detaches from the nuclear envelope in late prophase. We show that Scant creates a hyperactive form of Greatwall (Gwl) with altered specificity in vitro, another protein kinase recently implicated in mitotic entry in Drosophila and Xenopus. Excess Gwl activity in embryos causes developmental failure that can be rescued by increasing maternal Polo dosage, indicating that coordination between the two mitotic kinases is crucial for mitotic progression. Revertant alleles of Scant that restore fertility to polo–Scant heterozygous females are recessive alleles or deficiencies of gwl; they show chromatin condensation defects and anaphase bridges in larval neuroblasts. One recessive mutant allele specifically disrupts a Gwl isoform strongly expressed during vitellogenesis. Females hemizygous for this allele are sterile, and their oocytes fail to arrest in metaphase I of meiosis; both homologues and sister chromatids separate on elongated meiotic spindles with little or no segregation. This allelic series of gwl mutants highlights the multiple roles of Gwl in both mitotic and meiotic progression. Our results indicate that Gwl activity antagonizes Polo and thus identify an important regulatory interaction of the cell cycle

FlyTED: the Drosophila Testis Gene Expression Database

FlyTED, the Drosophila Testis Gene Expression Database, is a biological research database for gene expression images from the testis of the fruit fly Drosophila melanogaster. It currently contains 2762 mRNA in situ hybridization images and ancillary metadata revealing the patterns of gene expression of 817 Drosophila genes in testes of wild type flies and of seven meiotic arrest mutant strains in which spermatogenesis is defective. This database has been built by adapting a widely used digital library repository software system, EPrints (http://eprints.org/software/), and provides both web-based search and browse interfaces, and programmatic access via an SQL dump, OAI-PMH and SPARQL. FlyTED is available at http://www.fly-ted.org/

Inhibition of Ral GTPases Using a Stapled Peptide Approach

Aberrant Ras signalling drives numerous cancers and drugs to inhibit this are urgently required. This compelling clinical need, combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly and the focus has moved to the main downstream Ras-signalling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets, which were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development and there have therefore been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This therefore provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.This work was supported by a Cambridge Cancer Centre Pump Priming award to CA, DO and HRM, a BBSRC Studentship to NSC, and a National Institutes for Health grant (CA71443) and the Welch Foundation (grant number I-1414) to MAW.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via https://doi.org/10.1074/jbc.M116.72024

Roles for RNA export factor, Nxt1, in ensuring muscle integrity and normal RNA expression in Drosophila.

The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. Here we investigate the reduced viability of Nxt1 partial loss of function mutants, and describe a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. We detected differential expression of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in maintenance of muscle integrity. Our data also links the mRNA export pathway to a specific role in expression of mRNA and circRNA from common precursor genes, in vivo

Inhibition of Ral GTPases Using a Stapled Peptide Approach.

Aberrant Ras signaling drives numerous cancers, and drugs to inhibit this are urgently required. This compelling clinical need combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly, and the focus has moved to the main downstream Ras-signaling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets that were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development, and there have, therefore, been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical-stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This, therefore, provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.This work was supported by a Cambridge Cancer Centre Pump Priming award to CA, DO and HRM, a BBSRC Studentship to NSC, and a National Institutes for Health grant (CA71443) and the Welch Foundation (grant number I-1414) to MAW.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via https://doi.org/10.1074/jbc.M116.72024

Genome-wide analysis of gene regulation mechanisms during Drosophila spermatogenesis

Background During Drosophila spermatogenesis, testis-specific meiotic arrest complex (tMAC) and testis-specific TBP-associated factors (tTAF) contribute to activation of hundreds of genes required for meiosis and spermiogenesis. Intriguingly, tMAC is paralogous to the broadly expressed complex Myb-MuvB (MMB)/dREAM and Mip40 protein is shared by both complexes. tMAC acts as a gene activator in spermatocytes, while MMB/dREAM was shown to repress gene activity in many cell types. Results Our study addresses the intricate interplay between tMAC, tTAF, and MMB/dREAM during spermatogenesis. We used cell type-specific DamID to build the DNA-binding profiles of Cookie monster (tMAC), Cannonball (tTAF), and Mip40 (MMB/dREAM and tMAC) proteins in male germline cells. Incorporating the whole transcriptome analysis, we characterized the regulatory effects of these proteins and identified their gene targets. This analysis revealed that tTAFs complex is involved in activation of achi, vis, and topi meiosis arrest genes, implying that tTAFs may indirectly contribute to the regulation of Achi, Vis, and Topi targets. To understand the relationship between tMAC and MMB/dREAM, we performed Mip40 DamID in tTAF- and tMAC-deficient mutants demonstrating meiosis arrest phenotype. DamID profiles of Mip40 were highly dynamic across the stages of spermatogenesis and demonstrated a strong dependence on tMAC in spermatocytes. Integrative analysis of our data indicated that MMB/dREAM represses genes that are not expressed in spermatogenesis, whereas tMAC recruits Mip40 for subsequent gene activation in spermatocytes. Conclusions Discovered interdependencies allow to formulate a renewed model for tMAC and tTAFs action in Drosophila spermatogenesis demonstrating how tissue-specific genes are regulated