Christophe Roos

The Drosophila VEGF receptor homolog is expressed in hemocytes.

Several signalling pathways have been defined by studies of genes originally characterised in Drosophila. However, some mammalian signalling systems have so far escaped discovery in the fly. Here, we describe the identification and characterisation of fly homologs for the mammalian vascular endothelial growth factor/platelet derived growth factor (VEGF/PDGF) and the VEGF receptor. The Drosophila factor (DmVEGF-1) gene has two splice variants and is expressed during all stages, the signal distribution during embryogenesis being ubiquitous. The receptor (DmVEGFR) gene has several splice variants; the variations affecting only the extracellular domain. The most prominent form is expressed in cells of the embryonic haematopoietic cell lineage, starting in the mesodermal area of the head around stage 10 of embryogenesis. Expression persists in hemocytes as embryonic development proceeds and the cells migrate posteriorly. In a fly strain carrying a deletion uncovering the DmVEGFR gene, hemocytes are still present, but their migration is hampered and the hemocytes remain mainly in the anterior end close to their origin. These data suggest that the VEGF/PDGF signalling system may regulate the migration of the Drosophila embryonic haemocyte precursor cells.

The Drosophila hugin gene codes for myostimulatory and ecdysis-modifying neuropeptides

In a genomic screen we isolated the Drosophila gene hugin (hug, cytology 87C1-2) by cross-hybridisation to a human glial cell line-derived neurotrophic factor cDNA. Upon cDNA sequence analysis and in vitro expression assays, the hugin gene was found to encode a signal peptide containing proprotein that was further processed in Schneider-2 cells into peptides similar to known neuropeptides. Two of the peptides were similar to FXPRL-amides (pyrokinins) and to the ecdysis-triggering hormone, respectively. The former displayed myostimulatory activity in a bioassay on the cockroach hyperneural muscle preparation, as well as in the Drosophila heart muscle assay. Hugin is expressed during the later half of embryogenesis and during larval stages in a subgroup of neurosecretory cells of the suboesophageal ganglion. Ubiquitous ectopic hugin expression resulted in larval death predominantly at or shortly after ecdysis from second to third instar, suggesting that at least one of the posttranslational cleavage products affects molting of the larva by interfering with the regulation of ecdysis.

Activation of the nodA promoter by the nodD genes of Rhizobium galegae induced by synthetic flavonoids or Galega orientalis root exudate

Rhizobial nodD genes produce transcriptional regulators that, together with appropriate inducer compounds, activate the other symbiotic nodulation (nod) genes and initiate the nodule formation process. Two nodD homologues, nodD1 and nodD2, are present in the Rhizobium galegae strain HAMBI 1174. In this work we analysed their ability to induce the nodA promoter with synthetic inducers known to activate nod genes in other rhizobia. According to phylogenetic analysis, the inducer-specific carboxy-terminal part of the R. galegae nodD protein sequence groups together with those of Rhizobium leguminosarum and Sinorhizobium meliloti. However, the respective inducer compounds for their NodD proteins are not highly effective with R. galegae nodD products. The best inducer discovered with R. galegae nodD1 was the root exudate of the host plant of R. galegae, Galega orientalis. HPLC analyses revealed the presence of many divergent flavonoid compounds in the G. orientalis root exudate. The most effective HPLC fractions induced R. galegae nodD1 up to the level obtained by intact G. orientalis root exudate while apigenin and luteolin, which were also present in the root exudate, were only moderate inducers. A UV-Vis diode array spectrum of the most active peak indicated that the main inducer present in the G. orientalis root exudate is an unidentified chalcone-type compound. In the Galega-R. galegae interaction the first recognition between the NodD protein and the flavonoid inducer secreted from the roots of Galega is specific for these organisms, and thus partly responsible of the strict host specificity of this symbiosis.

Two otu transcripts are selectively localised in Drosophila oogenesis by a mechanism that requires a function of the otu protein.

The ovarian tumour gene (otu) is required for several processes during Drosophila oogenesis. The locus encodes two protein isoforms that have been proposed to act during different stages of oogenesis. Here we show that the corresponding otu mRNAs display a dynamic pattern of expression during oogenesis. The 4.1 kb mRNA encoding the 104 kDa isoform is expressed throughout adult oogenesis, but is mainly restricted to nurse cells. The 3.2 kb mRNA encoding the 98 kDa protein isoform is selectively localised in the oocyte up to stage 9. Both mRNAs are expressed abundantly in nurse cells at stages 10-11. We propose that the oocyte-specific function of otu is realised by the 98 kDa isoform. We show that the export of the 3.2 kb mRNA from the nurse cell nuclei requires a functional otu protein. The otu protein is also required for the correct distribution of the pumilio and oskar mRNAs, while the Bic-D, K10 and staufen mRNAs are localised in wild type fashion in otu mutants. Furthermore, we have observed a region of homology between the carboxy-terminal part of the otu protein and the mammalian microtubule associated proteins. The more severe the mutation in this region of homology, the more disturbed mRNA distribution is observed in otu mutants.

Nodulation Genes of Rhizobium Galegae

Rhizobium galegae infects goat’s rue (Galega orientalis and G. officinalis) in a very host specific manner. Nodules with an apical meristem are formed. R. galegae strains only form effective (nitrogen-fixing) nodules on their original host. Strains from G. orientalis infect G. officinalis, and give rise to almost fully developed nodules which fail to fix nitrogen, and vice versa (Lindstrom, 1989). G. orientalis is a new, perennial forage legume for Northern conditions. It is very persistent, resistant to disease and suitable for making silage. The plant and its rhizobia originate in the Caucasus and the plant requires inoculation with compatible rhizobia to grow well. Our laboratory has focused on the ecology of nitrogen fixation, the taxonomy of R. galegae, molecular biology of the interaction between symbionts and the development and application of molecular tools for identification and classification of the rhizobia. R. galegae is the only Rhizobium species described so far that belongs to the same phylogenetic branch as Agrobacterium vitis, A. rubi and biovar 1 (A. tumefaciens and A. radiobacter, Fig. 1). We are interested in the basis for the host specificity in our system and in the evolution of nodulation functions. This report describes the phylogeny of common nodulation genes and nodD genes of R. galegae and the identification of another nodulation gene region.

Analyses of the Drosophila quit, ovarian tumor and shut down mutants in oocyte differentiation using in situ hybridisation

We have studied the role of three loci, quit, ovarian tumor and shut down during oocyte differentiation in Drosophila by using in situ hybridisation and double mutant analyses. Mutations in qui and otu disturb the cystocyte divisions and the oocyte determination, while mutations in shu affect the cystocyte integrity, nevertheless allowing differentiation of normal-looking egg chambers with an oocyte. In all mutants the transport of molecules towards the posterior end of the egg chamber takes place as revealed by the accumulation of Bic-D or K10 transcripts. We show that the transport is ineffective in the qui and otu mutants apparently due to the lack of a properly differentiated oocyte. In the shu mutant the transport collapses and the oocyte is lost, leading to egg chambers with 15 nurse cells. We also show that one function of qui+ is to enhance otu+ mRNA expression, suggesting that these genes control the cystocyte maturation via the same pathway.

Polytene chromosomes show normal gene activity but some mRNAs are abnormally accumulated in the pseudonurse cell nuclei of Drosophila melanogaster otu mutants

Certain mutant alleles of the ovarian tumor (otu) locus give rise to polytene chromosomes in the pseudonurse cells (PNCs). We have previously shown that the banding pattern of these germ line-derived chromosomes is similar to that in the larval salivary gland chromosomes. In this study, we have examined the gene activity of these chromosomes. General gene expression from these chromosomes was studied by uridine autoradiography. The expression of specific genes was monitored by in situ hybridisation to mRNA and also by combining enhancer trap lines with otu mutants. We found that most of the genes studied were expressed in the PNCs as they were in the wild-type nurse cells. Four out of the 12 mRNAs studied accumulated in the nuclei instead of migrating to the cytoplasm. The intensity of accumulation directly correlated with the extent of polytenisation in the PNC nuclei. We suggest that the otu mRNA remains partly attached to the polytene chromosome template after transcription and discuss the effects of this phenomenon on polytenisation of the PNC chromosomes.

Drosophila non-muscle α-actinin is localized in nurse cell actin bundles and ring canals, but is not required for fertility

The single copy Drosophila alpha-actinin gene is alternatively spliced to generate three different isoforms that are expressed in larval muscle, adult muscle and non-muscle cells, respectively. We have generated novel alpha-actinin alleles, which specifically remove the non-muscle isoform. Homozygous mutant flies are viable and fertile with no obvious defects. Using a monoclonal antibody that recognizes all three splice variants, we compared alpha-actinin distribution in wild type and mutant embryos and ovaries. We found that non-muscle alpha-actinin was present in young embryos and in the embryonic central nervous system. In ovaries, non-muscle alpha-actinin was localized in the nurse cell subcortical cytoskeleton, cytoplasmic actin cables and ring canals. In the mutant, alpha-actinin expression remained in muscle tissues, but also in a subpopulation of epithelial cells in both embryos and ovaries. This suggests that various populations of non-muscle cells regulate alpha-actinin expression in different ways. We also show that ectopically expressed adult muscle-specific alpha-actinin localizes to all F-actin containing structures in the nurse cells in the absence of endogenous non-muscle alpha-actinin.