Second order regulator Collier directly controls intercalary-specific segment polarity gene expression

AbstractIn Drosophila, trunk metamerization is established by a cascade of segmentation gene activities: the gap genes, the pair rule genes, and the segment polarity genes. In the anterior head, metamerization requires also gap-like genes and segment polarity genes. However, because the pair rule genes are not active in this part of the embryo, the question on which gene activities are fulfilling the role of the second order regulator genes still remains to be solved. Here we provide first molecular evidence that the Helix–Loop–Helix–COE transcription factor Collier fulfills this role by directly activating the expression of the segment polarity gene hedgehog in the posterior part of the intercalary segment. Collier thereby occupies a newly identified binding site within an intercalary-specific cis-regulatory element. Moreover, we identified a direct physical association between Collier and the basic-leucine-zipper transcription factor Cap‘n'collar B, which seems to restrict the activating input of Collier to the posterior part of the intercalary segment and to lead to the attenuation of hedgehog expression in the intercalary lobes at later stages

A clustered set of three Sp-family genes is ancestral in the Metazoa: evidence from sequence analysis, protein domain structure, developmental expression patterns and chromosomal location

<p>Abstract</p> <p>Background</p> <p>The Sp-family of transcription factors are evolutionarily conserved zinc finger proteins present in many animal species. The orthology of the Sp genes in different animals is unclear and their evolutionary history is therefore controversially discussed. This is especially the case for the Sp gene <it>buttonhead </it>(<it>btd</it>) which plays a key role in head development in <it>Drosophila melanogaster</it>, and has been proposed to have originated by a recent gene duplication. The purpose of the presented study was to trace orthologs of <it>btd </it>in other insects and reconstruct the evolutionary history of the Sp genes within the metazoa.</p> <p>Results</p> <p>We isolated Sp genes from representatives of a holometabolous insect (<it>Tribolium castaneum</it>), a hemimetabolous insect (<it>Oncopeltus fasciatus</it>), primitively wingless hexapods (<it>Folsomia candida </it>and <it>Thermobia domestica</it>), and an amphipod crustacean (<it>Parhyale hawaienis</it>). We supplemented this data set with data from fully sequenced animal genomes. We performed phylogenetic sequence analysis with the result that all Sp factors fall into three monophyletic clades. These clades are also supported by protein domain structure, gene expression, and chromosomal location. We show that clear orthologs of the <it>D. melanogaster btd </it>gene are present even in the basal insects, and that the <it>Sp5</it>-related genes in the genome sequence of several deuterostomes and the basal metazoans <it>Trichoplax adhaerens </it>and <it>Nematostella vectensis </it>are also orthologs of <it>btd</it>.</p> <p>Conclusions</p> <p>All available data provide strong evidence for an ancestral cluster of three Sp-family genes as well as synteny of this Sp cluster and the Hox cluster. The ancestral Sp gene cluster already contained a <it>Sp5/btd </it>ortholog, which strongly suggests that <it>btd </it>is not the result of a recent gene duplication, but directly traces back to an ancestral gene already present in the metazoan ancestor.</p

Temporal and spatial control of transgene expression using laser induction of the hsp70 promoter

BACKGROUND: Precise temporal and spatial regulation of transgene expression is a critical tool to investigate gene function in developing organisms. The most commonly used technique to achieve tight control of transgene expression, however, requires the use of specific DNA enhancers that are difficult to characterize in non-model organisms. Here, we sought to eliminate the need for this type of sequence-based gene regulation and to open the field of functional genetics to a broader range of organisms. RESULTS: We have developed a new laser mediated method to heat shock groups of cells that provides precise spatio-temporal control of gene expression without requiring knowledge of specific enhancer sequences. We tested our laser-system in a transgenic line of Bicyclus anynana butterflies containing the EGFP reporter gene attached to the heat sensitive hsp70 promoter of Drosophila melanogaster. Whole organismal heat shocks demonstrated that this Drosophila promoter can drive gene expression in butterflies, and the subsequent laser heat shocks showed that it was possible to activate cell-specific gene expression in very precise patterns on developing pupal wings. CONCLUSION: This laser-mediated gene expression system will enable functional genetic investigations, i.e., the ectopic expression of genes and their knock-down in targeted groups of cells in model and non-model organisms with little or no available regulatory data, as long as a compatible heat-shock promoter is used and the target tissue is accessible to a laser beam. This technique will also be useful in evolutionary developmental biology as it will enable the study of the evolution of gene function across a variety of organisms

Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters

<p>Abstract</p> <p>Background</p> <p>The red flour beetle <it>Tribolium castaneum </it>has developed into an insect model system second only to <it>Drosophila</it>. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for <it>Tribolium </it>research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.</p> <p>Results</p> <p>Here we report the establishment of the GAL4/UAS binary expression system in <it>Tribolium castaneum</it>. Both GAL4Δ and GAL4VP16 driven by the endogenous heat shock inducible promoter of the <it>Tribolium hsp68 </it>gene are efficient in activating reporter gene expression under the control of the Upstream Activating Sequence (UAS). UAS driven ubiquitous tGFP fluorescence was observed in embryos within four hours after activation while <it>in-situ </it>hybridization against tGFP revealed expression already after two hours. The response is quick in relation to the duration of embryonic development in <it>Tribolium </it>- 72 hours with segmentation being completed after 24 hours - which makes the study of early embryonic processes possible using this system. By comparing the efficiency of constructs based on <it>Tribolium, Drosophila</it>, and artificial core promoters, respectively, we find that the use of endogenous core promoters is essential for high-level expression of transgenic constructs.</p> <p>Conclusions</p> <p>With the established GAL4/UAS binary expression system, ectopic misexpression approaches are now feasible in <it>Tribolium</it>. Our results support the contention that high-level transgene expression usually requires endogenous regulatory sequences, including endogenous core promoters in <it>Tribolium </it>and probably also other model systems.</p

Insulated piggyBac vectors for insect transgenesis

BACKGROUND: Germ-line transformation of insects is now a widely used method for analyzing gene function and for the development of genetically modified strains suitable for pest control programs. The most widely used transposable element for the germ-line transformation of insects is piggyBac. The site of integration of the transgene can influence gene expression due to the effects of nearby transcription enhancers or silent heterochromatic regions. Position effects can be minimized by flanking a transgene with insulator elements. The scs/scs' and gypsy insulators from Drosophila melanogaster as well as the chicken β-globin HS4 insulator function in both Drosophila and mammalian cells. RESULTS: To minimize position effects we have created a set of piggyBac transformation vectors that contain either the scs/scs', gypsy or chicken β-globin HS4 insulators. The vectors contain either fluorescent protein or eye color marker genes and have been successfully used for germ-line transformation of Drosophila melanogaster. A set of the scs/scs' vectors contains the coral reef fluorescent protein marker genes AmCyan, ZsGreen and DsRed that have not been optimized for translation in human cells. These marker genes are controlled by a combined GMR-3xP3 enhancer/promoter that gives particularly strong expression in the eyes. This is also the first report of the use of the ZsGreen and AmCyan reef fluorescent proteins as transformation markers in insects. CONCLUSION: The insulated piggyBac vectors should protect transgenes against position effects and thus facilitate fine control of gene expression in a wide spectrum of insect species. These vectors may also be used for transgenesis in other invertebrate species

Morphological and Transcriptomic Analysis of a Beetle Chemosensory System Reveals a Gnathal Olfactory Center

OR gene tissue expression and their chromosomal localization. a Venn diagram showing the number of ORs expressed (RPKM ≥ 0.5) in the different body parts: antennae, legs, mouthparts (as piece of the head capsule anterior of the antennae), heads (the whole head capsule including mouthparts but excluding the antennae), and bodies (excluding head and legs). b Venn diagram comparing our results (yellow, green) with data from Engsontia et al. [115] (blue, red). Number of expressed ORs, defined by RPKM ≥ 0.5 (yellow), by RT-PCR (blue), not expressed RPKM < 0.5 (green), or with no RT-PCR amplicon (red). ORs of the brown group were not previously tested by Engsontia et al. c Chromosomal localization of T. castaneum ORs. Based on the Georgia GA-2 strain genome assembly 3.0 [81], only chromosomal linkage groups containing an IR or SNMP are depicted. Gene clusters are indicated by a number referring to the chromosome and a letter conveys the relative position on the chromosome. The number of genes within this cluster is indicated in the square brackets. (PDF 277 kb

Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red flour beetle Tribolium castaneum, identifies embryonic lethal mutations and enhancer traps

<p>Abstract</p> <p>Background</p> <p>Given its sequenced genome and efficient systemic RNA interference response, the red flour beetle <it>Tribolium castaneum </it>is a model organism well suited for reverse genetics. Even so, there is a pressing need for forward genetic analysis to escape the bias inherent in candidate gene approaches.</p> <p>Results</p> <p>To produce easy-to-maintain insertional mutations and to obtain fluorescent marker lines to aid phenotypic analysis, we undertook a large-scale transposon mutagenesis screen. In this screen, we produced more than 6,500 new <it>piggyBac </it>insertions. Of these, 421 proved to be recessive lethal, 75 were semi-lethal, and eight indicated recessive sterility, while 505 showed new enhancer-trap patterns. Insertion junctions were determined for 403 lines and often appeared to be located within transcription units. Insertion sites appeared to be randomly distributed throughout the genome, with the exception of a preference for reinsertion near the donor site.</p> <p>Conclusion</p> <p>A large collection of enhancer-trap and embryonic lethal beetle lines has been made available to the research community and will foster investigations into diverse fields of insect biology, pest control, and evolution. Because the genetic elements used in this screen are species-nonspecific, and because the crossing scheme does not depend on balancer chromosomes, the methods presented herein should be broadly applicable for many insect species.</p

Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae)

<p>Abstract</p> <p>Background</p> <p>The sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly <it>Ceratitis capitata </it>(Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness.</p> <p>Results</p> <p>Here, we present a first alternative reproductive sterility system for medfly based on transgenic embryonic lethality. This system is dependent on newly isolated medfly promoter/enhancer elements of cellularization-specifically-expressed genes. These elements act differently in expression strength and their ability to drive lethal effector gene activation. Moreover, position effects strongly influence the efficiency of the system. Out of 60 combinations of driver and effector construct integrations, several lines resulted in larval and pupal lethality with one line showing complete embryonic lethality. This line was highly competitive to wildtype medfly in laboratory and field cage tests.</p> <p>Conclusion</p> <p>The high competitiveness of the transgenic lines and the achieved 100% embryonic lethality causing reproductive sterility without the need of irradiation can improve the efficacy of operational medfly SIT programs.</p

A conserved function of the zinc finger transcription factor Sp8/9 in allometric appendage growth in the milkweed bug Oncopeltus fasciatus

The genes encoding the closely related zinc finger transcription factors Buttonhead (Btd) and D-Sp1 are expressed in the developing limb primordia of Drosophila melanogaster and are required for normal growth of the legs. The D-Sp1 homolog of the red flour beetle Tribolium castaneum, Sp8 (appropriately termed Sp8/9), is also required for the proper growth of the leg segments. Here we report on the isolation and functional study of the Sp8/9 gene from the milkweed bug Oncopeltus fasciatus. We show that Sp8/9 is expressed in the developing appendages throughout development and that the downregulation of Sp8/9 via RNAi leads to antennae, rostrum, and legs with shortened and fused segments. This supports a conserved role of Sp8/9 in allometric leg segment growth. However, all leg segments including the claws are present and the expression of the leg genes Distal-less, dachshund, and homothorax are proportionally normal, thus providing no evidence for a role of Sp8/9 in appendage specification

Measuring multipartite entanglement via dynamic susceptibilities

Entanglement plays a central role in our understanding of quantum many body physics, and is fundamental in characterising quantum phases and quantum phase transitions. Developing protocols to detect and quantify entanglement of many-particle quantum states is thus a key challenge for present experiments. Here, we show that the quantum Fisher information, representing a witness for genuinely multipartite entanglement, becomes measurable for thermal ensembles via the dynamic susceptibility, i.e., with resources readily available in present cold atomic gas and condensed-matter experiments. This moreover establishes a fundamental connection between multipartite entanglement and many-body correlations contained in response functions, with profound implications close to quantum phase transitions. There, the quantum Fisher information becomes universal, allowing us to identify strongly entangled phase transitions with a divergent multipartiteness of entanglement. We illustrate our framework using paradigmatic quantum Ising models, and point out potential signatures in optical-lattice experiments.Comment: 5+5 pages, 3+2 figure