A Targeted Gain-of-Function Screen Identifies Genes Affecting Salivary Gland Morphogenesis/Tubulogenesis in Drosophila

During development individual cells in tissues undergo complex cell-shape changes to drive the morphogenetic movements required to form tissues. Cell shape is determined by the cytoskeleton and cell-shape changes critically depend on a tight spatial and temporal control of cytoskeletal behavior. We have used the formation of the salivary glands in the Drosophila embryo, a process of tubulogenesis, as an assay for identifying factors that impinge on cell shape and the cytoskeleton. To this end we have performed a gain-of-function screen in the salivary glands, using a collection of fly lines carrying EP-element insertions that allow the overexpression of downstream-located genes using the UAS-Gal4 system. We used a salivary-gland-specific fork head-Gal4 line to restrict expression to the salivary glands, in combination with reporters of cell shape and the cytoskeleton. We identified a number of genes known to affect salivary gland formation, confirming the effectiveness of the screen. In addition, we found many genes not implicated previously in this process, some having known functions in other tissues. We report the initial characterization of a subset of genes, including chickadee, rhomboid1, egalitarian, bitesize, and capricious, through comparison of gain- and loss-of-function phenotypes.     

Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange

Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.     

Meiosis in Drosophila melanogaster

Chromosome pairing during meiosis I in D. melanogaster males was investigated ultrastructurally by examining complete bivalents in electron micrographs of serial thin sections. The XY bivalent is characterized by the presence of unique material located between the two half-bivalents at the site of synapsis. The material has a fibrillar appearance and is less electron dense than the surrounding chromatin. YY bivalents in XYY males and XY bivalents containing the X chromosome, In(1)sc^4Lsc^8R, where the pairing sites of the X chromosome are inverted and partially deleted also possess this material. The material is not associated with autosomal bivalents and may represent a morphological manifestation of the hypothetical cohesive elements (collochores) which are thought to function in conjunction of the X and Y chromosomes (Cooper, 1964).     

Metabolic effects of CO2 anaesthesia in Drosophila melanogaster

Immobilization of insects is necessary for various experimental purposes, and CO₂ exposure remains the most popular anaesthetic method in entomological research. A number of negative side effects of CO₂ anaesthesia have been reported, but CO₂ probably brings about metabolic modifications that are poorly known. In this work, we used GC/MS-based metabolic fingerprinting to assess the effect of CO₂ anaesthesia in Drosophila melanogaster adults. We analysed metabolic variation of flies submitted to acute CO₂ exposure and assessed the temporal metabolic changes during short- and long-term recovery. We found that D. melanogaster metabotypes were significantly affected by the anaesthetic treatment. Metabolic changes caused by acute CO₂ exposure were still manifested after 14 h of recovery. However, we found no evidence of metabolic alterations when a long recovery period was allowed (more than 24 h). This study points to some metabolic pathways altered during CO₂ anaesthesia (e.g. energetic metabolism). Evidence of short-term metabolic changes indicates that CO₂ anaesthesia should be used with utmost caution in physiological studies when a short recovery is allowed. In spite of this, CO₂ treatment seems to be an acceptable anaesthetic method provided that a long recovery period is allowed (more than 24 h).     

Selection and recombination in Drosophila melanogaster

Summary Artificial selection for wing length in Drosophila melanogaster resulted in changed crossing-over frequencies between three marker genes on the 2nd chromosome, b, cn and vg.The results suggest that artificial selection is a causal agent in producing the observed changes; moreover it is suggested that the modifications in cross-over frequency are controlled by extra-nuclear factors.Research supported by C.N.R. Consiglio Nazionale delle Ricerche Roma, Grant n. 115.2298.4791.     

A Novel Cell Death Gene Acts to Repair Patterning Defects in Drosophila melanogaster

Cell death is a mechanism utilized by organisms to eliminate excess cells during development. Here, we describe a novel regulator of caspase-independent cell death, Mabiki (Mabi), that is involved in the repair of the head patterning defects caused by extra copies of bicoid in Drosophila melanogaster. Mabiki functions together with caspase-dependent cell death mechanisms to provide robustness during development.     

Evolutionary Rate Covariation Identifies New Members of a Protein Network Required for Drosophila melanogaster Female Post-Mating Responses

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP''s actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks.     

Regulation in rDNA-deficient Drosophila melanogaster

Bobbed mutants of Drosophila melanogaster were used to determine whether there is any functional compensation for deficiency in rDNA content. The rate of rRNA accumulation was measured in the testes of five bb mutants of different phenotypic severity and in a wild type strain. The different rates of rRNA accumulation were compared to the phenotype (macroscutellar bristle length) and found to have a direct correlation (as in Weinman, 1972). However, there was not a direct relationship between the rate of rRNA accumulation and rDNA content. It is concluded that there is regulation of rRNA accumulation in some mutants, but that the regulation cannot be considered to be a compensation for the lack of rDNA. These results are discussed relative to other observations on the regulation of RNA synthesis in Drosophila.     

Proteomics in Drosophila melanogaster.

To be able to understand cellular mechanisms, we require fully integrated data sets combining information about gene expression, protein expression, post-translational modification states, sub-cellular location and complex formation. Proteomics is a very powerful technique that can be applied to interrogate changes at the protein level. Studying this effectively requires specialised facilities within research institutes. Here, we describe the setting up and operation of such a facility, providing a resource for the Arabidopsis and Drosophila research communities.     

Tandem duplications in Drosophila melanogaster

In the tandem duplication Dp(1;1)Gr approximately one quarter of the euchromatic part of the X-chromosome is duplicated. Dp(1;1)Gr itself has no phenotypic effect, but it can be made visible by combining different alleles within the tandem duplication and the homologous X-chromosome. In heterozygous females crossing-over between the two X-chromosomes is strongly reduced while at the same time crossing-over in the distal regions of the autosomes is increased.