The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

Cordon-bleu is a conserved gene involved in neural tube formation

The axial midline is an important source of patterning and morphogenesis cues in the vertebrate embryo. The midline derives from a small group of cells in the gastrulating embryo, known as "the organizer" in recognition of its ability to organize an entire body plan. The mammalian organizer, the node, gives rise to axial midline structures: the notochord, dorsal foregut, and part of the floor plate of the neural tube. Only some of the genes that direct midline development are known. In this study, we present the complete coding sequence for a novel gene, cordon-bleu (cobl), expressed specifically in the node and its derivatives until organogenesis stages. The deduced sequence does not resemble any gene of known function. However, cobl is widely conserved: apparent orthologs and paralogs are found in many vertebrate species, with several sequence domains of high conservation but unknown function. We find that chicken cordon-bleu is similarly expressed in the node and its derivatives, suggesting functional conservation. We also report the sequence and nonoverlapping expression of a related mouse gene, Coblr1. Finally, we show that cobl interacts with the neurulation gene Vangl2 to facilitate midbrain neural tube closure, demonstrating roles for both cobl and Vangl2 in midbrain neurulation.     

WEB 2086, a platelet-activating factor antagonist, inhibits prophenoloxidase-activating system and hemocyte microaggregation reactions induced by Trypanosoma rangeli infection in Rhodnius prolixus hemolymph

The effects of the triazolodiazepine WEB 2086, a platelet-activating factor (PAF) antagonist, on hemocyte microaggregation and prophenoloxidase (proPO)-activating system in the hemolymph, hemocoelic infection and mortality in fifth-instar larvae of Rhodnius prolixus inoculated with Trypanosoma rangeli were investigated. Hemocoelic injection of short T. rangeli epimastigotes (1x10(4) parasites/insect) in R. prolixus that were previously fed with blood containing 1muM of WEB 2086 resulted in (i) reduced hemocyte microaggregations as well as an attenuated proPO system in the hemolymph and (ii) greater parasitemia and mortality among the insects. In vitro assays using hemolymph from insects previously fed with blood containing WEB 2086 exhibited attenuated hemocyte microaggregations when T. rangeli was employed as the inducer of the reaction, and this effect was not counteracted by PAF treatment. In vitro assays using hemolymph from insects previously fed with blood, regardless of WEB 2086 presence increased the PO activity when incubated with the parasites. However, the PO activity was drastically inhibited when hemolymph from insects fed with blood, whether or not it contained WEB 2086, was incubated with fat body homogenates from insects fed with blood containing WEB 2086. The addition of PAF did not enhance the PO activity. These analyses did not reveal any PAF influence on WEB 2086 effects in the two defense reactions.     

The Wiskott-Aldrich syndrome protein (WASP) is essential for myoblast fusion in Drosophila

In higher organisms, mononucleated myoblasts fuse to form multinucleated myotubes. During this process, myoblasts undergo specific changes in cell morphology and cytoarchitecture. Previously, we have shown that the actin regulator Kette (Hem-2/Nap-1) is essential for myoblast fusion. In this study, we describe the role of the evolutionary conserved Wiskott-Aldrich syndrome protein that serves as a regulator for the Arp2/3 complex for myoblast fusion. By screening an EMS mutagenesis collection, we discovered a new wasp allele that does not complete fusion during myogenesis. Interestingly, this new wasp3D3-035 allele is characterized by a disruption of fusion after precursor formation. The molecular lesion in this wasp allele leads to a stop codon preventing translation of the CA domain. Usually, the WASP protein exerts its function through the Arp2/3-interacting CA domain. Accordingly, a waspDeltaCA that is expressed in a wild-type background acts as dominant-negative during the fusion process. Furthermore, we show that the myoblast fusion phenotype of kette mutant embryos can be suppressed by reducing the gene dose of wasp3D3-035. Thus, Kette antagonizes WASP function during myoblast fusion.     

Caenorhabditis elegans dna-2 is involved in DNA repair and is essential for germ-line development

Caenorhabditis elegans germ cell proliferation and development were severely damaged in second generation dna-2 homozygotes. Even in the first generation, a much higher incidence of aberrant chromosomes in oocytes and resultantly higher embryonic lethality were found vs. wild type, when DNA breaks were induced by gamma-rays or camptothecin. The deficiency of dna-2 in combination with RNA interference on mre-11 gene expression synergistically aggravated germ-line development, especially oocyte formation. These results suggest that C. elegans Dna-2 is involved in a DNA repair pathway paralleling homologous recombination or non-homologous end joining with mre-11 participation.     

Effects of high temperature on the hemocyte cell cycle in silkworm larvae

To understand the inhibitory effects of high temperature on insect growth at the cellular level, we investigated the influence of high temperature on the proliferation and division of larval hemocytes in the silkworm, Bombyx mori. Although the total number of hemocytes in the larval body increased enormously over time at 26 degrees C, no increase was observed at 38 degrees C. The number of mitotic hemocytes in circulation increased between days 1 and 2 of the fourth larval stage at 26 degrees C, whereas fewer hemocytes were observed at 38 degrees C. Laser scanning cytometry revealed that the DNA content of hemocytes collected from the fourth-stadium larvae was predominantly 2C, 4C, and 8C, and the proportion of each type of hemocyte changed dynamically with development during the fourth instar. Specifically, the proportion of hemocytes with a higher DNA content increased gradually during the feeding phase then decreased during the molting phase at 26 degrees C; in contrast, no decrease was observed at 38 degrees C. The heat-induced accumulation of 8C hemocytes was mainly detected in granulocytes and plasmatocytes. These data suggest that high temperatures induce a G(2) arrest in larval hemocytes.     

Cybip,a starfish cyclin B-binding protein,is involved in meiotic M-phase exit

We designed a screen to identify starfish oocyte proteins able to bind monomeric cyclin B by affinity chromatography on a cyclin B splice variant displaying low affinity for cdc2. We identified a 15kDa protein previously described as a cdk-binding protein [Biochim. Biophys. Acta Mol. Cell Res. 1589 (2002) 219-231]. Cybip is encoded by a single polymorphic gene and the native protein is matured by cleaving a signal peptide. We firmly establish the fact that it is a true cyclin B-binding protein, since the recombinant protein binds recombinant cyclin B in absence of any cdk. Finally, we show that the microinjection of GST-cybip, and of anti-cybip antibody, in maturing starfish oocytes, inhibits H1 kinase and MPF inactivation, and first polar body emission.     

Drosophila crooked-neck protein co-fractionates in a multiprotein complex with splicing factors

The Drosophila crooked neck (crn) gene encodes an unusual TPR-containing protein whose function is essential for embryonic development. Homology with other TPR-proteins involved in cell cycle control, initially led to the proposal that Crn might play a critical role in regulation of embryonic cell divisions. Here, we show that Crn does not have a cell cycle function in the embryo. By using specific antibodies we also show that the Crn protein is a nuclear protein which localizes in "speckles" which could correspond to preferential localization of several other splicing factors. Fractionation of nuclear extracts on sucrose gradients revealed Crn in a 900 kDa multiproteic complex together with snRNPs, suggesting that Crn participates in the assembly of the splicing machinery in vivo.     

Abdominal-B is essential for proper sexually dimorphic development of the Drosophila gonad

Sexual dimorphism requires the integration of positional information in the embryo with the sex determination pathway. Homeotic genes are a major source of positional information responsible for patterning along the anterior-posterior axis in embryonic development, and are likely to play a critical role in sexual dimorphism. Here, we investigate the role of homeotic genes in the sexually dimorphic development of the gonad in Drosophila. We have found that Abdominal-B (ABD-B) is expressed in a sexually dimorphic manner in the embryonic gonad. Furthermore, Abd-B is necessary and sufficient for specification of a sexually dimorphic cell type, the male-specific somatic gonadal precursors (msSGPs). In Abd-B mutants, the msSGPs are not specified and male gonads now resemble female gonads with respect to these cells. Ectopic expression of Abd-B is sufficient to induce formation of extra msSGPs in additional segments of the embryo. Abd-B works together with abdominal-A to pattern the non-sexually dimorphic somatic gonad in both sexes, while Abd-B alone specifies the msSGPs. Our results indicate that Abd-B acts at multiple levels to regulate gonad development and that Abd-B class homeotic genes are conserved factors in establishing gonad sexual dimorphism in diverse species.     

Gurken, a TGF-alpha-like protein involved in axis determination in Drosophila, directly binds to the EGF-receptor homolog Egfr

The establishment of axial polarity in the Drosophila egg and embryo depends on intercellular communication between two cell types in the ovary, the germline, and the soma. The genes gurken and egfr encode two essential players of this communication pathway. Gurken protein, a TGF-alpha-like molecule, is expressed in the germline, while the EGF-receptor homolog, Egfr, is expressed in the somatic cells of the ovary. Using the yeast two-hybrid system we show here, for the first time, that Gurken protein directly binds to the extracellular domain of Egfr. This direct physical association requires the presence of an intact EGF motif within Gurken protein. Furthermore, we provide evidence that this characteristic motif may be sufficient for interaction with the receptor, at list in vitro. Our results firmly establish Gurken as the germline ligand of Drosophila Egfr.     

Dual functionality of O-GlcNAc transferase is required for Drosophila development

Post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) catalysed by O-GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein–protein interactions. Drosophila OGT (DmOGT) is encoded by super sex combs (sxc), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O-GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and DmOGT informed the rational design of DmOGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity.