Expression pattern of Drosophila translin and behavioral analyses of the mutant

Translin is an evolutionarily conserved approximately 27-kDa protein that binds to specific DNA and RNA sequences and has diverse cellular functions. Here, we report the cloning and characterization of the translin orthologue from the fruit fly Drosophila melanogaster. Under protein-denaturing conditions, purified Drosophila translin exists as a mixture of dimers and monomers just like human translin. In contrast to human translin, the Drosophila translin dimers do not appear to be stabilized by disulfide interactions. Drosophila translin shows a ubiquitous cytoplasmic localization in early embryonal syncytial stage, with an enhanced staining in ventral neuroblasts at later stages (8-9), which are probably at metaphase. An elevated expression was seen in several other cell types, such as cells around the tracheal pits in the embryo and oenocytes in the third instar larva. RNA in situ hybridization showed an increased expression in the ventral midline cells of the larval brain, suggesting a neuronal expression, which was corroborated by protein immunostaining. In adult flies, Drosophila translin is localized in the brain neuronal cell bodies and in early spermatocytes. Interestingly, Drosophila translin mutants exhibit an impaired motor response which is sex specific. Taken together, the multiple cellular localizations, the high neuronal expression and the attendant locomotor defect of the Drosophila translin mutant suggest that Drosophila translin may have roles in neuronal development and behavior analogous to that of mouse translin.     

Germ cells

Recently, cell signaling has been shown to be required for the formation of germ cells in the mouse embryo, direct observation of germ cell migration in living mouse embryos has been achieved, novel genes that control germ-cell migration have been identified in Drosophila, and the roles of many components of germ plasm in several species have become clearer.     

Genetic control of retinal specification and determination in Drosophila

The Drosophila compound eye has long served as an outstanding model system to study many processes, including cell fate specification, cell division, cell growth and cell death. In addition, exploring the molecular basis of eye specification in Drosophila has identified a set of nuclear factors that trigger the conversion of a group of multipotent epithelial cells into eye primordia. These nuclear factors act in complex networks to regulate retinal specification and appear to be conserved throughout phylogeny. Finally, evidence suggests that these nuclear networks have been co-opted to specify cell fates in other tissues. We review the latest developments in the field of retinal specification in Drosophila and discuss several future directions that remain open for investigation.     

Larval RNAi in Drosophila?

RNA interference (RNAi) has become a common method of gene knockdown in many model systems. To trigger an RNAi response, double-stranded RNA (dsRNA) must enter the cell. In some organisms such as Caenorhabditis elegans, cells can take up dsRNA from the extracellular environment via a cellular uptake mechanism termed systemic RNAi. However, in the fruit fly Drosophila melanogaster, it is widely believed that cells are unable to take up dsRNA, although there is little published data to support this claim. In this study, we set out to determine whether this perception has a factual basis. We took advantage of traditional Gal4/upstream activation sequence (UAS) transgenic flies as well as the mosaic analysis with a repressible cell marker (MARCM) system to show that extracellular injection of dsRNA into Drosophila larvae cannot trigger RNAi in most Drosophila tissues (with the exception of hemocytes). Our results show that this is not due to a lack of RNAi machinery in these tissues as overexpression of dsRNA inside the cells using hairpin RNAs efficiently induces an RNAi response in the same tissues. These results suggest that, while most Drosophila tissues indeed lack the ability to uptake dsRNA from the surrounding environment, hemocytes can initiate RNAi in response to extracellular dsRNA. We also examined another insect, the red flour beetle Tribolium castaneum, which has been shown to exhibit a robust systemic RNAi response. We show that virtually all Tribolium tissues can respond to extracellular dsRNA, which is strikingly different from the situation in Drosophila. Our data provide specific information about the tissues amenable to RNAi in two different insects, which may help us understand the molecular basis of systemic RNAi.     

Asymmetric cell division

Asymmetric cell division is a conserved mechanism for partitioning information during mitosis. Over the past several years, significant progress has been made in our understanding of how cells establish polarity during asymmetric cell division and how determinants, in the form of localized proteins and mRNAs, are segregated. In particular, genetic studies in Drosophila and Caenorhabditis elegans have linked cell polarity, G protein signaling and regulation of the cytoskeleton to coordination of mitotic spindle orientation and localization of determinants. Also, several new studies have furthered our understanding of how asymmetrically localized cell fate determinants, such as the Numb, a negative regulator Notch signaling, functions in biasing cell fates in the developing nervous system in Drosophila. In vertebrates, analysis of dividing neural progenitor cells by in vivo imaging has raised questions about the role of asymmetric cell divisions during neurogenesis.     

Mouse germ cell clusters form by aggregation as well as clonal divisions

After their arrival in the fetal gonad, mammalian germ cells express E-cadherin and are found in large clusters, similar to germ cell cysts in Drosophila. In Drosophila, germ cells in cysts are connected by ring canals. Several molecular components of intercellular bridges in mammalian cells have been identified, including TEX14, a protein required for the stabilization of intercellular bridges, and several associated proteins that are components of the cytokinesis complex. This has led to the hypothesis that germ cell clusters in the mammalian gonad arise through incomplete cell divisions. We tested this hypothesis by generating chimeras between GFP-positive and GFP-negative mice. We show that germ cell clusters in the fetal gonad arise through aggregation as well as cell division. Intercellular bridges, however, are likely restricted to cells of the same genotype.     

Conservation of epigenetic regulation, ORC binding and developmental timing of DNA replication origins in the genus Drosophila

There is much interest in how DNA replication origins are regulated so that the genome is completely duplicated each cell division cycle and in how the division of cells is spatially and temporally integrated with development. In the Drosophila melanogaster ovary, the cell cycle of somatic follicle cells is modified at precise times in oogenesis. Follicle cells first proliferate via a canonical mitotic division cycle and then enter an endocycle, resulting in their polyploidization. They subsequently enter a specialized amplification phase during which only a few, select origins repeatedly initiate DNA replication, resulting in gene copy number increases at several loci important for eggshell synthesis. Here we investigate the importance of these modified cell cycles for oogenesis by determining whether they have been conserved in evolution. We find that their developmental timing has been strictly conserved among Drosophila species that have been separate for approximately 40 million years of evolution and provide evidence that additional gene loci may be amplified in some species. Further, we find that the acetylation of nucleosomes and Orc2 protein binding at active amplification origins is conserved. Conservation of DNA subsequences within amplification origins from the 12 recently sequenced Drosophila species genomes implicates members of a Myb protein complex in recruiting acetylases to the origin. Our findings suggest that conserved developmental mechanisms integrate egg chamber morphogenesis with cell cycle modifications and the epigenetic regulation of origins.     

Telomere loss in somatic cells of Drosophila causes cell cycle arrest and apoptosis

Checkpoint mechanisms that respond to DNA damage in the mitotic cell cycle are necessary to maintain the fidelity of chromosome transmission. These mechanisms must be able to distinguish the normal telomeres of linear chromosomes from double-strand break damage. However, on several occasions, Drosophila chromosomes that lack their normal telomeric DNA have been recovered, raising the issue of whether Drosophila is able to distinguish telomeric termini from nontelomeric breaks. We used site-specific recombination on a dispensable chromosome to induce the formation of a dicentric chromosome and an acentric, telomere-bearing, chromosome fragment in somatic cells of Drosophila melanogaster. The acentric fragment is lost when cells divide and the dicentric breaks, transmitting a chromosome that has lost a telomere to each daughter cell. In the eye imaginal disc, cells with a newly broken chromosome initially experience mitotic arrest and then undergo apoptosis when cells are induced to divide as the eye differentiates. Therefore, Drosophila cells can detect and respond to a single broken chromosome. It follows that transmissible chromosomes lacking normal telomeric DNA nonetheless must possess functional telomeres. We conclude that Drosophila telomeres can be established and maintained by a mechanism that does not rely on the terminal DNA sequence.     

Proliferative control in Drosophila stem cells

The relationship between cell growth (cell mass increase over time) and cell division is poorly understood in animal stem cells. Recent studies in several Drosophila stem cell types have provided the tools to interrogate this relationship. In several cases (brat, mei-P26, pros, bam, lethal giant larvae, polo), mutations have been defined that trigger tumorous overproliferation of progenitor cells and reveal how unrestricted self-renewing capacity is controlled. Moreover, microRNAs have been discovered as essential regulators of stem cell division rate and identity, suggesting that stem cell self-renewal depends on protein translational control. Biosynthetic capacity has also been found to be limiting for stem cell division rates. Finally, asymmetric cell division can impose dominant differentiation signals in a stem cell's daughter, and this can inhibit the stem cell-specific proliferation signature and lock in cell cycle exit.     

Established cell lines ofDrosophila hydei

Summary Four cell lines have been isolated fromDrosophila hydei embryos. Three lines have a normal XY karyotype, the fourth has an XO karyotype with an additional small heterochromatic fragment. The cells contain presumable cytoplasmic virus like particles. This work was supported by a travel grant to Dr. N. H. Lubsen from the Netherlands Organization for the Advancement of Pure Research (Z. W. O.).     

Loss of the tumor suppressor Pten promotes proliferation of Drosophila melanogaster cells in vitro and gives rise to continuous cell lines

In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (Ras(V12)) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing Ras(V12). Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines.     

Cell ablation by ectopic expression of cell death genes, ced-3 and Ice, in Drosophila

We have developed a system for killing specific cells in Drosophila using ectopic expression of cell death genes. CED-3 and ICE (caspase-1) are proteins required for programmed cell death in the nematode Caenorhabditis elegans and in mammals, respectively. Our previous study has shown that both ced-3 and Ice can elicit cell death in Drosophila . By expressing ced-3 or Ice in several kinds of cells using a GAL4-UAS system and examining the resulting morphological defects, we show that these abnormalities are thought to be caused by the action of ced-3 or Ice genes. As cells are killed by apoptosis in our system, we could eliminate the possibility of harmful effects on the neighboring cells. Our system provides an alternative and novel cell ablation method to elucidate mechanisms of cell differentiation and cell-cell interactions during development in Drosophila .     

Mass Determination of the Unit Cell of the Innermost Chorionic Layer in Drosophilidae by Scanning Transmission Electron Microscopy

The innermost chorionic layer (ICL) in eggshells of Drosophila melanogaster is a naturally occurring patchwork of thin three-dimensional crystalline plates located between the inner endochorion and the vitelline envelope. The mass-per-unit area of the ICL has been measured from scanning transmission electron microscope images of isolated unstained material and it was possible to distinguish up to four layers with the majority of the crystalline sheets being one to three layers thick. Taking into account the unit cell areas for the different crystals, we have estimated the mean ICL subunit sizes to be 36 kDa for Drosophila melanogaster, 35 kDa for Drosophila auraria, and 33 kDa for Drosophila teissieri. The results suggest that the three different Drosophilidae species have very similar average subunit masses.     

In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [^3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [³H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [³H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.     

A Drosophila model to study the functions of TWIST orthologs in apoptosis and proliferation

The twist gene has been characterized for its role in myogenesis in several species. In addition, in mammalian cultured cells, it has been shown that twist is a potential oncogene antagonizing p53-dependent apoptosis. To study, in vivo, the role of twist in apoptosis and proliferation, we constructed transgenic Drosophila lines allowing ectopic expression of different twist orthologs. We report that: (i) Drosophila twist induces apoptosis and activates the reaper promoter, (ii) nematode twist induces arrest of proliferation without apoptosis, and (iii) human twist retains its potentialities observed in mammalian cultured cells and antagonizes Drosophila p53-dependent apoptosis. In addition, we show that human twist is able to induce cell proliferation in Drosophila. Data suggest that the pathway by which human twist antagonizes Drosophila p53 could be conserved. These transgenic lines thus constitute a powerful tool to identify targets and modifiers of human twist.     

Phylogenetic conservation of the regulatory and functional properties of the Vav oncoprotein family

Vav proteins are phosphorylation-dependent GDP/GTP exchange factors for Rho/Rac GTPases. Despite intense characterization of mammalian Vav proteins both biochemically and genetically, there is little information regarding the conservation of their biological properties in lower organisms. To approach this issue, we have performed a characterization of the regulatory, catalytic, and functional properties of the single Vav family member of Drosophila melanogaster. These analyses have shown that the intramolecular mechanisms controlling the enzyme activity of mammalian Vav proteins are already present in Drosophila, suggesting that such properties have been set up before the divergence between protostomes and deuterostomes during evolution. We also show that Drosophila and mammalian Vav proteins have similar catalytic specificities. As a consequence, Drosophila Vav can trigger oncogenic transformation, morphological change, and enhanced cell motility in mammalian cells. Gain-of-function studies using transgenic flies support the implication of this protein in cytoskeletal-dependent processes such as embryonic dorsal closure, myoblast fusion, tracheal development, and the migration/guidance of different cell types. These results highlight the important roles of Vav proteins in the signal transduction pathways regulating cytoskeletal dynamics. Moreover, they indicate that the foundations for the regulatory and enzymatic activities of this protein family have been set up very early during evolution.