Browsing by Author "Hansen, Dave D."
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Item Open Access The C. elegans Sex-determining Gene fem-2 Encodes a Putative Protein Phosphatase(The American Society for Cell Biology, 1995) Pilgrim, Dave; McGregor, Angela; Jackle, Petra; Johnson, Troy; Hansen, Dave D.; Biological Sciences; Faculty of Sciencedetermination in Caenorhabditis elegans suggests that the gene fem-2 plays an important role in regulating a pathway transducing a non-cell-autonomous signal to a nuclear transcription factor. The wild-type fem-2 gene was cloned by identifying sequences from the C. elegans physical map that could restore normal Fem-2 function to homozygous mutant fem-2 transgenic animals. cDNA sequences mapping to the minimal rescuing region correspond to an open reading frame with a sequence similar to protein phosphatase 2C enzymes from systems as diverse as yeast, humans, and plants, but the alignments suggest that FEM-2 falls into a separate class of proteins than the canonical homologues. Several fem-2 mutant alleles were sequenced, and the mutations are predicted to cause protein changes consistent with their observed phenotypes, such as missense mutations in conditional alleles, and a nonsense mutation in a predicted null allele. This is the first evidence implicating phosphorylation and/or dephosphorylation as a control mechanism in C. elegans sex determinationItem Open Access Caenorhabditis elegans atx-2 Promotes Germline Proliferation and the Oocyte Fate(Genetics Society of America, 2004-10) Maine, Eleanor M.; Hansen, Dave D.; Springer, Deborah; Vought, Valarie E.; Biological Sciences; Faculty of Science; University of CalgaryIn the Caenorhabditis elegans germline, proliferation is induced by Notch-type signaling. Entry of germ cells into meiosis is triggered by activity of the GLD-1 and GLD-2 pathways, which function redundantly to promote meiosis and/or inhibit proliferation. Activation of the germline Notch-type receptor, GLP-1, ultimately inhibits the activities of the GLD-1 and GLD-2 pathways. We previously identified several ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we show that atx-2 is an ego gene. Our data suggest that ATX-2 is not a positive regulator of the GLP-1 signaling pathway and GLP-1 signaling is not the sole positive regulator of ATX-2 activity. Moreover, our data indicate that GLP-1 must have an additional function, which may be to repress activity of a third meiotic entry pathway that would work in parallel with the GLD-1 and GLD-2 pathways. In addition to its role in proliferation, ATX-2 acts downstream of FOG-2 to promote the female germline fate.Item Open Access Control of the proliferation versus meiotic development decision in the C. elegansgermline through regulation of GLD-1 protein accumulation(The Company of Biologists 2004, 2003) Hansen, Dave D.; Wilson-Berry, Laura; Dang,Thanh; Schedl, Tim; Biological Sciences; Faculty of Science; University of CalgaryMaintenance of the stem cell population in the C. elegans germline requires GLP-1/Notch signaling. We show that this signaling inhibits the accumulation of the RNA binding protein GLD-1. In a genetic screen to identify other genes involved in regulating GLD-1 activity, we identified mutations in the nos-3 gene, the protein product of which is similar to the Drosophila translational regulator Nanos. Our data demonstrate that nos-3 promotes GLD-1 accumulation redundantly with gld-2, and that nos-3 functions genetically downstream or parallel to fbf, an inhibitor of GLD-1 translation. We show that the GLD-1 accumulation pattern is important in controlling the proliferation versus meiotic development decision, with low GLD-1 levels allowing proliferation and increased levels promoting meiotic entry.Item Open Access Epsin potentiates Notchpathway activity in Drosophilaand C. elegans(The Company of Biologists 2004, 2004) Tian, Xiaolin; Hansen, Dave D.; Schedl, Tim; Skeath, James B.; Biological Sciences; Faculty of Science; University of Calgarypathway are known to modulate the activity of different signaling pathways. Epsins promote endocytosis and are postulated to target specific proteins for regulated endocytosis. Here, we present a functional link between the Notch pathway and epsins. We identify the Drosophila ortholog of epsin, liquid facets (lqf), as an inhibitor of cardioblast development in a genetic screen for mutants that affect heart development. We find that lqf inhibits cardioblast development and promotes the development of fusion-competent myoblasts, suggesting a model in which lqf acts on or in fusion-competent myoblasts to prevent their acquisition of the cardioblast fate. lqf and Notch exhibit essentially identical heart phenotypes, and lqf genetically interacts with the Notch pathway during multiple Notch-dependent events in Drosophila. We extended the link between the Notch pathway and epsin function to C. elegans, where the C. elegans lqf ortholog acts in the signaling cell to promote the glp-1/Notch pathway activity during germline development. Our results suggest that epsins play a specific, evolutionarily conserved role to promote Notch signaling during animal development and support the idea that they do so by targeting ligands of the Notch pathway for endocytosis.Item Open Access Evolution of the PP2C Family in Caenorhabditis: Rapid Divergence of the Sex-Determining Protein FEM-2(Springer-Verlag, 2002) Stothard, Paul; Hansen, Dave D.; Pilgrim, Dave; Biological Sciences; Faculty of Science; University of CalgaryTo investigate the causes and functional significance of rapid sex-determining protein evolution we compared three Caenorhabditis elegans genes encoding members of the protein phosphatase 2C (PP2C) family with their orthologs from another Caenorhabditis species (strain CB5161). One of the genes encodes FEM-2, a sex-determining protein, while the others have no known sex-determining role. FEM-2’s PP2C domain was found to be more diverged than the other PP2C domains, supporting the notion that sex-determining proteins are subjected to selective pressures that allow for or cause rapid divergence. Comparison of the positions of amino acid substitutions in FEM-2 with a solved three-dimensional structure suggests that the catalytic face of the protein is highly conserved among C. elegans, CB5161, and another closely related species C. briggsae. However, the non-conserved regions of FEM-2 cannot be said to lack functional importance, since fem-2 transgenes from the other species were unable to rescue the germ-line defect caused by a C. elegans fem-2 mutation. To test whether fem-2 functions as a sex-determining gene in the other Caenorhabditis species we used RNA-mediated interference (RNAi). fem-2 (RNAi) in C. elegans and C. briggsae caused germ-line feminization, but had no noticeable effect in CB5161. Thus the function of fem-2 in CB5161 remains uncertain.Item Open Access GERp95, a Membrane-associated Protein that Belongs to a Family of Proteins Involved in Stem Cell Differentiation(The American Society for Cell Biology, 1999) Cikaluk, Darren E.; Tahbaz, Nasser; Hendricks, Linda C.; DiMattia, Gabriel E.; Hansen, Dave D.; Pilgrim, Dave; Hobman, Tom C.; Biological Sciences; Faculty of Science; University of CalgaryA panel of mAbs was elicited against intracellular membrane fractions from rat pancreas. One of the antibodies reacted with a 95-kDa protein that localizes primarily to the Golgi complex or the endoplasmic reticulum (ER), depending on cell type. The corresponding cDNA was cloned and sequenced and found to encode a protein of 97.6 kDa that we call GERp95 (Golgi ER protein 95 kDa). The protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as signal peptides or transmembrane domains. Biochemical analysis suggests that GERp95 is a cytoplasmically exposed peripheral membrane protein that exists in a protease-resistant complex. GERp95 belongs to a family of highly conserved proteins in metazoans and Schizosaccharomyces pombe. It has recently been determined that plant and Drosophila homologues of GERp95 are important for controlling the differentiation of stem cells (Bohmert et al., 1998; Cox et al., 1998; Moussian et al., 1998). In Caenorhabditis elegans, there are at least 20 members of this protein family. To this end, we have used RNA interference to show that the GERp95 orthologue in C. elegans is important for maturation of germ-line stem cells in the gonad. GERp95 and related proteins are an emerging new family of proteins that have important roles in metazoan development. The present study suggests that these proteins may exert their effects on cell differentiation from the level of intracellular membranes.Item Open Access Identification of genes expressed in the hermaphrodite germ line of C. elegans using SAGE(BioMed Central, 2009-05) Wang, Xin; Zhao, Yongjun; Wong, Kim; Ehlers, Peter; Kohara, Yuji; Jones, Steven J; Marra, Marco A; Holt, Robert A; Moerman, Donald G; Hansen, Dave D.Item Open Access Molecular Evolution of a Sex Determination Protein: FEM-2 (PP2C) in Caenorhabditis(Genetics Society of America, 1998-04) Hansen, Dave D.; Pilgrim, Dave; Biological Sciences; Faculty of Science; University of CalgarySomatic sex determination in Caenorhabditis elegans involves a signal transduction pathway linking a membrane receptor to a transcription factor. The fem-2 gene is central to this pathway, producing a protein phosphatase (FEM-2) of the type 2C (PP2C). FEM-2 contains a long amino terminus that is absent in canonical PP2C enzymes. The function of this domain is difficult to predict, since it shows no sequence similarity to any other known proteins or motifs. Here we report the cloning of the fem-2 homologue from Caenorhabditis briggsae (Cb-fem-2). The sequence identity is much higher than that observed for other C. briggsae homologues of C. elegans sex determination proteins. However, this level is not uniform across the entire lengths of the proteins; it is much lower in the amino termini. Thus, the two domains of the same protein are evolving at different rates, suggesting that they have different functional constraints. Consistent with this, Cb-FEM-2 is able to replace some, but not all, of the Ce-FEM-2 in vivo function. We show that removal of the amino terminus from Ce-FEM-2 has no effect on its in vitro phosphatase activity, or its ability to replace the in vivo function of a yeast PP2C enzyme, but that it is necessary for proper FEM-2 function in worms. This demonstrates that the amino terminus is not an extended catalytic domain or a direct negative regulator of phosphatase activity.Item Open Access Multi-pathway control of the proliferation versus meiotic development decision in the Caenorhabditis elegans germline(Elsevier, 2004) Hansen, Dave D.; Hubbard, E. Jane Albert; Schedl, Tim; Biological Sciences; Faculty of Science; University of CalgaryAn important event in the development of the germline is the initiation of meiotic development. In Caenorhabditis elegans, the conserved GLP-1/Notch signaling pathway regulates the proliferative versus meiotic entry decision, at least in part, by spatially inhibiting genes in the gld-1 and gld-2 parallel pathways, which are proposed to either inhibit proliferation and/or promote meiotic development. Mutations that cause constitutive activation of the GLP-1 pathway, or inactivation of both the gld-1 and gld-2 parallel pathways, result in a tumorous germline in which all cells are thought to be proliferative. Here, to analyze proliferation and meiotic entry in wild-type and mutant tumorous germlines, we use anti-REC-8 and anti-HIM-3 specific antibodies as markers, which under our fixation conditions, stain proliferative and meiotic cells, respectively. Using these makers in wild-type animals, we find that the border of the switch from proliferation to meiotic entry is staggered in late-larval and adult germlines. In wild-type adults, the switch occurs between 19 and 26 cell diameters from the distal end, on average. Our analysis of mutants reveals that tumorous germlines that form when GLP-1 is constitutively active are completely proliferative, while tumors due to inactivation of the gld-1 and gld-2 pathways show evidence of meiotic entry. Genetic and time course studies suggest that a third pathway may exist, parallel to the GLD-1 and GLD-2 pathways, that promotes meiotic development.