Understanding gene function and genetic relationships is elemental in our efforts to better understand biological systems. Here, I describe a reliable high-throughput approach, Systematic Genetic Interaction analysis (SGI), capable of revealing both weak and strong genetic interactions in the nematode Caenorhabditis elegans. I also present evidence that UNC-73 functions cell-autonomously in an UNC-40 pathway to direct muscle arm extension in C. elegans. Previous efforts to systematically describe genetic interactions between redundant genes on a global scale either have focused on core biological processes in protozoans or have surveyed catastrophic interactions in metazoans. I investigated synthetic genetic interactions between eleven ‘query’ mutants in conserved signal transduction pathways and hundreds of ‘target’ genes compromised by RNAi. A network of 1246 genetic interactions was uncovered through an unbiased global analysis of the interaction matrix, establishing the largest metazoan genetic interaction network to date. To investigate how genetic interactions connect genes on a systems-wide level, the SGI network was superimposed with existing networks of physical, genetic, phenotypic and co-expression interactions. Fifty-six putative functional modules were identified within the superimposed network, one of which regulates fat accumulation and is coordinated by bar-1(ga80)/β-catenin interactions. This led to the discovery that SGI interactions link distinct functional modules on a global scale, which is a previously unappreciated level of organization within metazoan systems. In addition, I present evidence that the properties of genetic networks are conserved between C. elegans and S. cerevisiae, but that the connectivity of the interactions within the current networks is not. Although the buffering between functional modules may differ between species, studying these differences may provide insight into the evolution of divergent form and function. In C. elegans the postsynaptic membrane of the neuromuscular junction reaches its destination through an active process of guided cell extension. The worm has 95 body wall muscles (BWMs) that extend projections called 'muscle arms' to motor axons. The muscle arms harbour the postsynaptic elements of neuromuscular junctions. The stereotypical pattern of muscle arm extension was exploited in a forward genetic screen for new genes required for guided cell migration by looking for mutations that caused a reduction in the number of arms that extend to the motor axons. One of the resulting mutants was tr117, which extended half the number of arms compared to wild type animals. Genetic mapping, complementation tests, and sequencing revealed that tr117 was a mutation in unc-73/Trio, which encodes a guanine nucleotide exchange factor. Expression of UNC-73 specifically in the BWMs rescued the muscle arm development defects of unc-73(e936) mutants, indicating that UNC-73 functions cell-autonomously to regulate muscle arm extension. UNC-73::CFP was localized to muscle arm termini in a pattern similar to that of UNC-40/Dcc, which directs muscle arm extension. UNC-73 over-expression suppressed the Madd phenotype of unc-40 null worms and unc-73(e936) suppressed ectopic myopodia induced by UNC-40 over-expression. These results indicate that UNC-73 functions downstream of UNC-40 in a pathway that regulates muscle arm extension.

The anti-nematodal drug ivermectin hyperactivates invertebrate-specific glutamate-gated chloride channels (GluCls) causing pharyngeal paralysis and a cessation of feeding and growth. I find that for C. elegans even brief exposure to ivermectin can lead to irreversible pharyngeal paralysis. Ivermectin induces heterogeneous vacuolation in the pharynx that appears slowly and accumulates over several days. This vacuolation is almost completely rescued by a mutation in avr-15, which codes for the alpha-subunit of pharyngeal GluCls. The vacuoles stain strongly with Lysotracker Red and are therefore likely to be acidic compartments of the endosomal-lysosomal system. Examination of mutants defective for endocytosis (rme-1, rme-8, and cup-5) uncovers the presence of acidic vacuoles identical in appearance to ivermectin-induced vacuoles. Further, RME-1, a marker for recycling endosomes, is shown to redistribute soon after ivermectin exposure. Examination of the effects of ivermectin on extrapharyngeal neurons expressing ectopic avr-15 reveals an apoptotic phenotype that is shown to be ced-independent.

dsc-4(qm182), a mutation that suppresses the lengthened defecation cycle of clk-1 also suppresses the delay in germline development. dsc-4 encodes a putative orthologue of microsomal triglyceride transfer protein (MTP), a protein essential for the assembly and secretion of apo-B-containing low density lipoproteins (LDL). The effect of dsc-4 on clk-1(qm30), coupled to studies of apoB homologues in worms led to a model suggesting the possibility of using C. elegans in the study of LDL-like lipoprotein particles. The impact of the level of lipoproteins is particularly evident in the germline developmental rate of the worms. / We report here a further elucidation of clk-1 mutants in the study of the biology of LDL-like particles. In particular, we investigated the effect of targeting LDL receptor-like genes by RNA interference (RNAi) on the egg laying rate of clk-1(qm30). We find positive modulating effects by disruption of these putative LDL receptors. In confirmation of our model of lipoprotein metabolism in clk-1 mutants, we find that disruption of these putative LDL receptors produces strikingly different effects in wild-type, clk-1(qm30) or clk-1(qm30); dsc-4(qm182) animals. / In addition, we report unexpected effects of various clk-1 alleles on the phenotypes of animals in which lrp-1 and rme-2 are disrupted. Specifically, we observe an allele specific amelioration of the phenotypes associated with disruption of these genes (abnormal molting and sterility, respectively). We discuss the possible significance of these findings. (Abstract shortened by UMI.)

Serotonin is a very important neuromodulatory agent that affects many physiological and behavioral responses of both vertebrates and invertebrates. In helminths, especially parasitic ones, not much is known about the biosynthesis and mode of action of serotonin or any of the related biogenic amine neurotransmitters, such as catecholamines (dopamine and noradrenaline). In this study, we cloned two full length cDNAs from Schistosoma mansoni encoding tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH). TPH and TH catalyze the rate limiting steps in the biosynthesis of serotonin and catecholamines, respectively. Both enzymes were expressed in Escherichia coli and the purified proteins were shown to have TPH and TH activities. This indicates that S. mansoni, and possibly other parasitic helminths, may be capable of synthesizing serotonin and catecholamines endogenously. In the second part of our studies, we looked at the mode of action of serotonin in helminths, in particular the molecular properties of serotonergic G protein-coupled receptors (GPCR). We cloned two helminth GPCRs, one from the free living nematode Caenorhabditis elegans and the second from S. mansoni. The C. elegans receptor (5-HT2Ce) was shown to encode a functional serotonin receptor with structural and signaling properties similar to those of mammalian 5-HT2 receptors. However, its agonist I antagonist binding profile differed from previously characterized serotonin receptors. The cloned S. mansoni receptor (SmGPCRx) was found to represent a new structural class of receptor, which shared about the same level of amino acid sequence homology with various biogenic amines receptors, such as serotonin, catecholamines, and octopamine receptors. Additional sequence analysis and immunolocalization studies confirmed that SmGPCRx possesses structural characteristics of a GPCR. SmGPCRx is the first GPCR ever cloned from a parasitic flatworm. Taken together, these studies mark an important first step to

Understanding gene function and genetic relationships is elemental in our efforts to better understand biological systems. Here, I describe a reliable high-throughput approach, Systematic Genetic Interaction analysis (SGI), capable of revealing both weak and strong genetic interactions in the nematode Caenorhabditis elegans. I also present evidence that UNC-73 functions cell-autonomously in an UNC-40 pathway to direct muscle arm extension in C. elegans. Previous efforts to systematically describe genetic interactions between redundant genes on a global scale either have focused on core biological processes in protozoans or have surveyed catastrophic interactions in metazoans. I investigated synthetic genetic interactions between eleven ‘query’ mutants in conserved signal transduction pathways and hundreds of ‘target’ genes compromised by RNAi. A network of 1246 genetic interactions was uncovered through an unbiased global analysis of the interaction matrix, establishing the largest metazoan genetic interaction network to date. To investigate how genetic interactions connect genes on a systems-wide level, the SGI network was superimposed with existing networks of physical, genetic, phenotypic and co-expression interactions. Fifty-six putative functional modules were identified within the superimposed network, one of which regulates fat accumulation and is coordinated by bar-1(ga80)/β-catenin interactions. This led to the discovery that SGI interactions link distinct functional modules on a global scale, which is a previously unappreciated level of organization within metazoan systems. In addition, I present evidence that the properties of genetic networks are conserved between C. elegans and S. cerevisiae, but that the connectivity of the interactions within the current networks is not. Although the buffering between functional modules may differ between species, studying these differences may provide insight into the evolution of divergent form and function. In C. elegans the postsynaptic membrane of the neuromuscular junction reaches its destination through an active process of guided cell extension. The worm has 95 body wall muscles (BWMs) that extend projections called 'muscle arms' to motor axons. The muscle arms harbour the postsynaptic elements of neuromuscular junctions. The stereotypical pattern of muscle arm extension was exploited in a forward genetic screen for new genes required for guided cell migration by looking for mutations that caused a reduction in the number of arms that extend to the motor axons. One of the resulting mutants was tr117, which extended half the number of arms compared to wild type animals. Genetic mapping, complementation tests, and sequencing revealed that tr117 was a mutation in unc-73/Trio, which encodes a guanine nucleotide exchange factor. Expression of UNC-73 specifically in the BWMs rescued the muscle arm development defects of unc-73(e936) mutants, indicating that UNC-73 functions cell-autonomously to regulate muscle arm extension. UNC-73::CFP was localized to muscle arm termini in a pattern similar to that of UNC-40/Dcc, which directs muscle arm extension. UNC-73 over-expression suppressed the Madd phenotype of unc-40 null worms and unc-73(e936) suppressed ectopic myopodia induced by UNC-40 over-expression. These results indicate that UNC-73 functions downstream of UNC-40 in a pathway that regulates muscle arm extension.

During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1::gfp transgene cyIs4. The VC motor neurons are ventrally located neurons that extend two processes. VC1, VC2, VC3, and VC6 extend axons along the anterior-posterior (A/P) axis; VC4 and VC5 extend axons around the vulva along a mediolateral left-right (L/R) axis perpendicular to the A/P axis. We identified and showed that vang-1/Van Gogh, a core component of planar cell polarity (PCP) signalling pathway, acts cell-autonomously in VC4 and VC5 neurons and non-autonomously from the epithelial cells to restrict neurite formation along the A/P axis. vang-1 mutant animals display ectopic neurites along the A/P axis. Using a candidate gene approach, we further identified and revealed two additional core members of PCP signalling, Prickle (PRKL-1) and Dishevelled (DSH-1), to play a role in A/P-directed neurite suppression. We also showed prkl-1 and dsh-1 genetically interact with vang-1 and VANG-1 is required to suppress A/P-directed neurite outgrowth from larval stage 4 to adulthood. Overexpression of VANG-1 results in a loss-of-function (lof) phenotype, suggesting that an appropriate level of VANG-1 activity is important. Additionally, vang-1/prkl-1, and dsh-1 may interact in parallel pathways. Our findings implicate PCP genes to play a previously unidentified role in maintaining polarized neuronal morphology by inhibiting neuronal outgrowth responses to environmental cues.

The glutamate-gated chloride (GluCl) channel family of receptors are members of the ligand-gated ion channel (LGIC) family. In addition to being essential for physiological and behavioural aspects of an organism, they are also exploited as a target for the drug ivermectin. A novel GluCl subunit in C. elegans, GLC-4, was characterized in order to further understand the diversity of GluCls and their implications in biological and behavioural processes. Sequencing of cDNAs generated by RT-PCR indicate that GLC-4 possesses the typical features of a GluCl subunit. In addition, they also suggest possible alternative splicing of glc-4 resulting in a slightly truncated transcript. A glc-4 mutant, glc-4(ok212), was used to investigate the role of GLC-4 in C. elegans ivermectin sensitivity and behaviour. Glc-4(ok212) worms were found to be hypersensitive to low concentrations of ivermectin and experienced a slight hyperreversal behaviour. Genetic, molecular, and electrophysiological evidence is also provided suggesting an interaction between GLC-4 and AVR-14, another GluCl subunit in C. elegans. We hypothesize a direct association between GLC-4 and AVR-14 which together form a heteromeric channel in vivo .

Dissertation (Ph.D.)--University of Toledo, 2007. / Typescript. "Submitted as partial fulfillment of the requirements for The Doctor of Philosophy iin Biology." Bibliography: leaves 96-105.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 107-114). Also available for download via the World Wide Web; free to University of Oregon users.