Johannes Bischof

An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases

Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a φC31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the φC31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the φC31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, high-throughput screening of large cDNA sets and regulatory elements.

A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila

Overexpression screens are used to explore gene functions in Drosophila, but this strategy suffers from the lack of comprehensive and systematic fly strain collections and efficient methods for generating such collections. Here, we present a strategy that could be used efficiently to generate large numbers of transgenic Drosophila strains, and a collection of 1149 UAS-ORF fly lines that were created with the site-specific ΦC31 integrase method. For this collection, we used a set of 655 genes that were cloned as two variants, either as an open reading frame (ORF) with a native stop codon or with a C-terminal 3xHA tag. To streamline the procedure for transgenic fly generation, we demonstrate the utility of injecting pools of plasmids into embryos, each plasmid containing a randomised sequence (barcode) that serves as a unique identifier for plasmids and, subsequently, fly strains. We also developed a swapping technique that facilitates the rapid exchange of promoters and epitope tags in vivo, expanding the versatility of the ORF collection. The work described here serves as the basis of a systematic library of Gal4/UAS-regulated transgenes.

Bozozok directly represses bmp2b transcription and mediates the earliest dorsoventral asymmetry of bmp2b expression in zebrafish

Formation of the gastrula organizer requires suppression of ventralizing signals and, in fish and frog, the need to counteract the effect of ubiquitously present maternal factors that activate the expression of Bmps. How the balance between dorsalizing and ventralizing factors is shifted towards organizer establishment at late blastula stages is not well understood. Mutations in zebrafish bozozok (boz) cause severe defects in axial mesoderm and anterior neurectoderm and affect organizer formation. The boz gene encodes the homeodomain protein Bozozok/Dharma and its expression in the region of the organizer is activated through β-catenin signaling. Here, we investigate the molecular mechanism by which boz contributes to the establishment of the organizer. We demonstrate that the homeodomain protein Boz acts as a transcriptional repressor in zebrafish: overexpression of an En-Boz fusion protein can rescue the boz phenotype, whereas a VP16-Boz fusion protein acts as an antimorph. Expression analysis of bmp2b indicates that Boz negatively regulates bmp2b in the prospective organizer. We demonstrate that this Boz activity is independent of that of other zygotic genes, because it also occurs when translation of zygotic genes is suppressed by cycloheximide (CHX). We identify two high-affinity binding sites for Boz within the first intron of the bmp2b gene. Deletion of these control elements abolishes Boz-dependent repression of bmp2b in the early blastula. Thus, Boz directly represses bmp2b by binding to control elements in the bmp2b locus. We propose that early transcriptional repression of bmp2b by Boz is one of the first steps toward formation of a stable organizer, whereas the later-acting Bmp antagonists (e.g. Chordin, Noggin) modulate Bmp activity in the gastrula to induce patterning along the dorsoventral axis. Thus, similar to Drosophila Dpp, asymmetry of Bmp expression in zebrafish is initiated at the transcriptional level, and the shape of the gradient and its function as a morphogen are later modulated by post-transcriptional mechanisms.

Recombinases and their use in gene activation, gene inactivation, and transgenesis.

The site-specific recombinase FLP is used in Drosophila to precisely manipulate the genome, in particular, to eliminate gene function by mitotic recombination and to activate transgenes in discrete populations of cells. These approaches are already part of the standard tool kit for studying gene function. The number of applications for the FLP recombinase has increased over the years and further members of the large family of site-specific recombinases are being added to the arsenal of fly geneticists, most recently, the phiC31 integrase. This chapter will introduce these recombinases and describe how such instruments are utilized to accurately manipulate the Drosophila genome.

Coop functions as a corepressor of Pangolin and antagonizes Wingless signaling

Wingless (Wg) signaling regulates expression of its target genes via Pangolin and Armadillo, and their interacting cofactors. In the absence of Wg, Pangolin mediates transcriptional repression. In the presence of Wg, Pangolin, Armadillo, and a cohort of coactivators mediate transcriptional activation. Here we uncover Coop (corepressor of Pan) as a Pangolin-interacting protein. Coop and Pangolin form a complex on DNA containing a Pangolin/TCF-binding motif. Overexpression of Coop specifically represses Wg target genes, while loss of Coop function causes derepression. Finally, we show that Coop antagonizes the binding of Armadillo to Pangolin, providing a mechanism for Coop-mediated repression of Wg target gene transcription.

Generation of a transgenic ORFeome library in Drosophila

Overexpression screens can be used to explore gene function in Drosophila melanogaster, but to demonstrate their full potential, comprehensive and systematic collections of fly strains are required. Here we provide a protocol for high-throughput cloning of Drosophila open-reading frames (ORFs) that are regulated by upstream activation sequences (UAS sites); the resulting GAL4-inducible UAS-ORF plasmid library is then used to generate Drosophila strains by ΦC31 integrase–mediated site-specific integration. We also provide details for FLP/FRT-mediated in vivo exchange of epitope tags (or regulatory regions) in the ORF library strains, which further extends the potential applications of the library. These transgenic UAS-ORF strains are a useful resource to complement and validate genetic experiments performed with loss-of-function mutants and RNA interference (RNAi) lines. The duration of the complete protocol strongly depends on the number of ORFs required, but embryos can be injected and balanced fly stocks can be established within ∼7–8 weeks for a few genes.

Drosophila β-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

Cells in ectotherms function normally within an often wide temperature range. As temperature dependence is not uniform across all the distinct biological processes, acclimation presumably requires complex regulation. The molecular mechanisms that cope with the disruptive effects of temperature variation are still poorly understood. Interestingly, one of five different β-tubulin paralogs, βTub97EF, was among the genes upregulated at low temperature in cultured Drosophila cells. As microtubules are known to be cold sensitive, we analyzed whether βTub97EF protects microtubules at low temperatures. During development at the optimal temperature (25°C), βTub97EF was expressed in a tissue-specific pattern primarily in the gut. There, as well as in hemocytes, expression was increased at low temperature (14°C). Although βTub97EF mutants were viable and fertile at 25°C, their sensitivity within the well-tolerated range was slightly enhanced during embryogenesis specifically at low temperatures. Changing β-tubulin isoform ratios in hemocytes demonstrated that β-Tubulin 97EF has a pronounced microtubule stabilizing effect. Moreover, βTub97EF is required for normal microtubule stability in the gut. These results suggest that βTub97EF upregulation at low temperature contributes to acclimation by stabilizing microtubules. Highlighted Article: Ectotherms thrive within an often remarkable temperature range. At low temperatures, βTub97EF, a β-tubulin paralog stabilizing microtubules, is upregulated in a tissue-specific manner in Drosophila melanogaster.

Generation of a versatile BiFC ORFeome library for analyzing protein-protein interactions in live Drosophila

Transcription factors achieve specificity by establishing intricate interaction networks that will change depending on the cell context. Capturing these interactions in live condition is however a challenging issue that requires sensitive and non-invasive methods. We present a set of fly lines, called ‘multicolor BiFC library’, which covers most of the Drosophila transcription factors for performing Bimolecular Fluorescence Complementation (BiFC). The multicolor BiFC library can be used to probe two different binary interactions simultaneously and is compatible for large-scale interaction screens. The library can also be coupled with established Drosophila genetic resources to analyze interactions in the developmentally relevant expression domain of each protein partner. We provide proof of principle experiments of these various applications, using Hox proteins in the live Drosophila embryo as a case study. Overall this novel collection of ready-to-use fly lines constitutes an unprecedented genetic toolbox for the identification and analysis of protein-protein interactions in vivo.