Thompson, Owen et al. (2013) International Worm Meeting "The Million Mutation Project and Beyond."

Strasbourger, Pnina, Adair, Ryan, Ewing, Brent, Turner, Emily, Chaudhry, Iasha, Hillier, LaDeana, Moerman, Donald G., Fernando, Lisa, Shendure, Jay, Shen, Bin, Au, Vinci, Hutter, Harald, Lau, Joanne, Miller, Angela, Thompson, Owen, Edgley, Mark, Waterston, Robert H., Taylor, Jon, Raymant, Greta, Flibotte, Stephane

[International Worm Meeting, 2013]

We have created a library of 2,007 mutagenized C. elegans strains, each sequenced to a target depth of 15X coverage, to provide the research community with mutant alleles for each of the worm's more than 20,000 genes. The library contains over 820,000 unique SNVs with an average of eight non-synonymous changes per gene and more than 16,000 indel and copy number changes, providing an unprecedented genetic resource for C. elegans. We supplemented this collection with 40 sequenced wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets shows that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. Comparison with expected events suggests that very few missense alleles are incompatible with life but more than 40% are subject to long term selection. All the strains are available through the CGC; all the sequence changes have been deposited in WormBase and our own interactive web site (http://genome.sfu.ca/mmp/about.html). Loss-of-function alleles are now available for 13,760 of 20,514 protein coding genes. To identify mutations in the remaining genes, including essential genes, we use the following strategy. F1 progeny of mutagenized P0s are minimally propagated before sequencing and analysis. We use gain or loss of restriction sites to facilitate following the mutations through subsequent steps to isolate homozygous viable animals, or to create heterozygous balanced lethals. Pilot experiments have demonstrated the feasibility of this approach; we have identified new nonsense alleles of known essential genes, as well as nonsense and splicing mutations in several genes with no previous null mutations.

Thompson A et al. (1997) International C. elegans Meeting "A SCREEN FOR NEW GENES IN THE C. ELEGANS VULVAL DEVELOPMENT PATHWAY"

Miller LM, Thompson A

[International C. elegans Meeting, 1997]

We have used a unique screen in an attempt to identify new genes involved in vulval development. Previous genetic screens by other groups for vulvaless (Vul) mutations that prevent proper development of the vulva have identified genes required for generation of vulval precursor cells, specification of vulval cell fates, or execution of those cell fates. These direct screens for Vul mutations are powerful, however, this type of screen is likely to be saturated. In fact, the most common mutations identified in the direct screen are alleles of genes involved in the specification of vulval cell fates, the most well-understood aspect of vulval development. To avoid isolating alleles of specification genes, we screened for vul mutations in a lin-31 mutant background. lin-31 encodes a transcription factor required for the proper specification of vulval cell fates. Since the lin-31 mutant phenotype is epistatic to the Vul phenotype of the specification genes, this screen will not identify specification genes and will instead identify generation genes or execution genes downstream of lin-31. In this study, animals carrying a null mutation in lin-31 were mutagenized and the progeny from 8,500 F2 clones were screened for plates containing nearly 100% Vul animals. This screen yielded 30 candidates One mutation, lm25, is particularly interesting. The lin-31;lm25 strain is 57% Muv, 40% Vul, 43% sterile, and 22% Pvul (protruding vulva). lin-31 mutant strains without the lm25 mutation are 77% Muv, 10-15% Vul, 0% sterile, and less than 5% Pvul. Preliminary lineage analysis of lm25 suggests possible lineage and/or morphogenesis defects in the vulval precursor cells. The fact that lm25 displays a low penetrance for both the Vul and the Pvul phenotypes indicates that it may not have been identified in previous Vul or Pvul screens and may therefore represent a new gene. lm92 and lm97 are also likely to represent new genes, because their Vul phenotypes are not present in a wild-type lin-31 background.

Thompson, Josh et al. (2015) International Worm Meeting "Mechanisms of SYS-1/beta-catenin centrosomal localization in early embryonic blastomeres."

Thompson, Josh, Vora, Setu, Phillips, Bryan

[International Worm Meeting, 2015]

Asymmetric cell division, the unequal distribution of cell fate determinants between daughter cells, is a critical system underlying the development and maintenance of the varied tissue types of a mature organism. Despite widespread utilization of such divisions, the mechanisms behind induction of asymmetry are less understood due to its complex, pleiotropic, and often functionally redundant effects. Throughout C. elegans development, Wnt/beta-catenin signaling induces asymmetric distribution of Wnt signaling components to polarize a mother cell, differentially regulating Wnt target genes in its daughter cells. For instance, a proper Wnt signaling response results in accumulation of cytoplasmic SYS-1/beta-catenin in the proximal 'signaled' cell, which, along with co-activator POP-1/TCF, activates genes to induce the corresponding cell fate. Despite its asymmetric regulation immediately post-division, cytoplasmic SYS-1 localizes symmetrically to mitotic centrosomes. Recent data from our lab indicate that centrosomal localization of SYS-1 serves as a clearance mechanism to increase the robustness of Wnt-mediated polarity. However, the method of SYS-1 trafficking to and accumulation at the centrosome is unknown. In the early embryo, centrosomal localization of the b-catenin SYS-1 is dependent on the centrosomal scaffolding protein RSA-2. When SYS-1 is prevented from loading to the centrosome, by loss of RSA-2, it co-localizes to the region of kinetochore microtubules. This localization suggests a directed transport of SYS-1 to centrosomes along microtubules via the action of molecular motors. We conducted an RNAi and chemical screen specifically aimed at depleting microtubules and microtubule transport proteins and quantified resulting SYS-1 centrosomal accumulation. We focused on dynein and dynactin components as the primary motor machinery for the observed minus end trafficking. We identified several dynein subunits by RNAi knockdown that are required for proper centrosomal localization of embryonic SYS-1/beta-catenin. Further, the preliminary data from microtubule-destabilizing and ATP-depleting chemical treatment indicate that both ATP and microtubules are important for SYS-1 centrosomal localization. Together, these results begin to characterize a regulatory role for the multimeric dynein/dynactin cargo binding complex in SYS-1/beta-catenin localization.

Thompson FJ et al. (1998) European Worm Meeting "Comparison of cytidine deaminase in Brugia pahangi and C. elegans"

Devaney E, Hunter SJ, Thompson FJ

[European Worm Meeting, 1998]

A differential screen of a post-infective L3 cDNA library resulted in the isolation of the Brugia homologue of cytidine deaminase (cdd), This enzyme is involved in the salvage pathway for pyrimidine nucleosides and has been identified as a site-specific RNA editing enzyme in mammals. Recombinant Brugia cdd was expressed and used in a variety of assays. The Brugia cdd bound a synthetic RNA template but was unable to edit. The expression pattern of cdd was investigated by Northern blotting and by semi-quantitative RT-PCR. This analysis revealed that the cdd was highly expressed in the post-infective L3 and L4 but barely detectable in adult parasites. Recently both the cdd homologues (cel 1 and cel 2) from C. elegans have been cloned and the expression pattern of these genes throughout the life cycle analysed. Cel 2 expression was shown to peak once at the end of each larval stage and the transcript was also identified in the adult worm following embryogenesis. We aim to localise the expression of this molecule in elegans following micoinjection of reporter constructs and, using RNA interference assays, we hope to define the function of cdd in the nematode.

Fiona J Thompson et al. (2002) European Worm Meeting "Genome analysis of the effect of the host immune response on Strongyloides ratti."

Fiona J Thompson, Brandi Chiapelli, Claire Murphy, Clare P Wilkes, Mark E Viney, Jim McCarter, Sandra Clifton

[European Worm Meeting, 2002]

The establishment, survival and fecundity of nematodes is dramatically affected by the host immune response. However the molecular and biochemical nature of these effects have yet to be fully explored. We propose to exploit the nematode genome sequencing projects and EST data to analyse these phenomena. To this end we have generated a number of cDNA libraries from different stages of Strongyloides ratti including those from parasitic adults which had been exposed to different degrees of immune pressure. To date 7,793 ESTs have been sequenced and the cluster analysis of these clones will be presented. In order to generate microarrays all unique clones are being amplifed by PCR and arrayed. These microarrays will be differentially probed with cDNA isolated from populations of worms from varying immune pressures. We aim to identify gene sets whose expression levels vary as a result of changes in the host immune response and as such may be the basis of the immune dependant changes in the fitness of nematode infections.

F Thompson et al. (1999) International C. elegans Meeting "Comparison of Cytidine Deaminase in Brugia pahangi and C. elegans"

E Devaney, F Thompson, C Britton, S Hunter

[International C. elegans Meeting, 1999]

Lymphatic filarial worms are nematode parasites which cause disease in many tropical regions. These parasites have a developmental cycle which involves both an insect vector and a mammalian host. The filarial homologue of cytidine deaminase (CDD) was cloned in a differential screen which sought to identify cDNAs which were up-regulated as the L3 of Brugia transfers between hosts. CDDs are a family of enzymes involved in the salvage pathway for pyrimidine nucleosides and the site-specific editing of mRNA in mammals. In Brugia , the expression of cdd is triggered by exposure to the mammalian environment and is largely restricted to the post-infective larval stages. This expression pattern is not consistent with that of a house keeping gene. Recombinant Brugia CDD was expressed and shown to possess the characteristics of a functional CDD enzyme. In a heterologous editing assay the recombinant protein was shown to exhibit a novel RNA binding activity but was unable to edit the RNA sequence. In order to determine the functional significance of these molecules in nematodes we have begun to characterise the CDDs in C. elegans . Two C.elegans homologues, Ce-cdd-1 and Ce-cdd-2 were identified. These genes have been cloned and the expression pattern throughout the life-cycle analysed. As in Brugia , the mRNAs are expressed in a stage-specific manner. Reporter constructs have been made and will be used to define the temporal and spatial expression patterns of these genes. Using RNA interference assays, a reproducible phenotype has been obtained; the worms are longer and thinner than wild type and display a head-lifting motion. RNA binding and editing assays using recombinant Ce -CDD-1 and Ce -CDD-2 are in progress. A recent search of ACeDB using a Prosite signature for the cytidine deaminase active site identified a third potential CDD on cosmid C29F5. The predicted protein contains an additional amino acid residue in the active site which may affect its catalytic activity. However the predicted size of this protein (31kDa) and the presence of several motifs which are all characteristics of the mammalian editing enzymes warrants further study of this gene.

Thompson-Peer, Katherine L. et al. (2011) International Worm Meeting "HBL-1 patterns synaptic remodeling in C. elegans."

Thompson-Peer, Katherine L., Kaplan, Joshua, Bai, Jihong

[International Worm Meeting, 2011]

Synapses are added and removed extensively during the development of the nervous system, and to a lesser extent throughout the lifetime of an animal. Although synaptic refinement plays a pivotal role in establishing circuit and likely cognitive function in many if not most systems, little is known about the genetic pathways governing this process. In C. elegans, the GABAergic DD motor neurons undergo developmentally programmed remodeling, whereby synapses onto ventral body muscles are eliminated and replaced with dorsal synapses. We show that DD remodeling is extensively patterned and we identify the transcription factor HBL-1 as a central regulator of this process. Remodeling is restricted to the DD neurons by UNC-55, which represses hbl-1 expression in the GABAergic VD neurons, explaining the cell-specificity of remodeling. Mutants lacking HBL-1 have delayed DD remodeling, whereas precocious remodeling is observed in mutants lacking the microRNA mir-84, which inhibits translation of the hbl-1 mRNA. Finally, we observe that DD remodeling is activity-dependent. Moreover, bidirectional changes in network activity alter the transcription of hbl-1, and adjust the timing of remodeling in an HBL-1-dependent manner. Thus microRNA, network activity, and transcriptional regulatory pathways converge on hbl-1 to pattern DD plasticity.

Beth E Thompson et al. (2003) International Worm Meeting "Genetic control of early germline proliferation in C. elegans"

Beth E Thompson, Judith Kimble, Andrei G Petcherski

[International Worm Meeting, 2003]

During larval development, germ cells divide from the two present at hatching to about a thousand in adults by a variable pattern of divisions to generate. These germline divisions are controlled by a combination of Notch signaling and RNA regulation. The fbf genes, fbf-1 and fbf-2, encode members of the Puf family of RNA binding proteins (Wickens et al 2002). fbf-1 fbf-2 double mutants proliferate normally during early larval stages, but all germ cells enter into meiosis during L4 and differentiate as sperm (Crittenden et al 2002). We have found that two other genes, fog-1 and fog-3, can be critical for germline divisions during L1 and L2. FOG-1 is a CPEB homologue required for the sperm fate (Barton and Kimble 1991; Jin et al, 2001), and FOG-3 is a Tob family protein also required the sperm fate (Ellis and Kimble, 1995; Chen et al 2000). The germline proliferates normally in both fog-1 and fog-3 single mutants, but they make no sperm and instead make oocytes continuously. However, in an fbf-1 fbf-2 double mutant background, a reduction of either fog-1 or fog-3 by RNAi results in a germline with a reduced number of germ cells compared to the fbf-1 fbf-2 double mutant. Most commonly there appear to be only 4-16 cells; these cells appear to be oocytes; no cell death has been observed. We are currently building the fog-1; fbf-1 fbf-2 triple mutant to characterize these defects in more depth. Progress will be reported at the meeting. Wickens et al (2002) Trends in Genetics 18(3), 150-157; Crittenden et al (2002) Nature 417, 660-663; Barton and Kimble (1991) Genetics 125, 29-39; Jin et al (2001) Developmental Biology 229, 537-553; Ellis and Kimble (1995) Genetics 139, 561-577; Chen et al (2000) Developmental Biology 217, 77-90

Beth Thompson et al. (2002) Mid-west Worm Meeting "Setting up to identify FBF target mRNAs in C. elegans"

Judith Kimble, Beth Thompson

[Mid-west Worm Meeting, 2002]

FBF is an RNA binding protein that is essential for regulation of multiple germline fates. Two nearly identical genes, fbf-1 andfbf-2, code for the largely redundant FBF-1 and FBF-2 proteins. An fbf-1 fbf-2 double mutant makes only sperm and lacks germline stem cells in late development (Crittenden et al., 2002). To date, only two target mRNAs are well established (Zhang et al., 1997; Crittenden et al., 2002). To identify additional targets, we will use the approach successfully pioneered by Lee and Schedl (2001) to find GLD-1 target mRNAs. Specifically, we will generate transgenic worms expressing epitope-tagged versions of FBF-1 and FBF-2 to co-precipitate FBF associated mRNAs. At the current time, we have obtained rescue of fbf-1 fbf-2 double mutants with a genomic fragment of fbf-1 and a distinct genomic fragment bearing fbf-2. The line bearing the fbf-2 transgene has been stable for over 45 generations. Approximately 510% of the animals are fertile, and remaining animals are sterile. We are currently making the epitope-tagged versions, and will report progress at the meeting.

Thompson, Joshua W. et al. (2017) International Worm Meeting "Mechanisms of SYS-1/beta-catenin centrosomal localization in early embryonic blastomeres."

Thompson, Joshua W., Vora, Setu M., Phillips, Bryan T.

[International Worm Meeting, 2017]

Asymmetric cell division, the unequal distribution of cell fate determinants between daughter cells, is a critical system underlying the development and maintenance of varied tissue types in a multicellular organism. Despite widespread utilization of such divisions, the mechanisms responsible for induction of asymmetry are less understood due to the complex, pleiotropic, and often functionally redundant signaling systems involved. Throughout C. elegans development, the Wnt/beta-catenin asymmetry pathway induces the asymmetric distribution of Wnt signaling components to polarize a mother cell and therefore differentially regulate Wnt target genes in its daughter cells. Despite this asymmetry in its regulation immediately post-division, cytoplasmic SYS-1 localizes symmetrically to mitotic centrosomes in a manner consistent with well conserved beta-catenin localization. The subfunctionalization of C. elegans' 4 beta-catenin homologs allows us to investigate the role of this phenomenon with particular focus on the transcriptional regulation of the Wnt/ beta -catenin asymmetry pathway. Recent data indicate that centrosomal localization of SYS-1 serves as a clearance mechanism to increase the robustness of Wnt-mediated polarity. However, the method and regulation of SYS-1 trafficking to and accumulation at the centrosome is unknown. In the early embryo, this centrosomal localization of the SYS-1 is dependent on the centrosomal scaffolding protein RSA-2. Loss of RSA-2 by RNAi prevents SYS-1 centrosomal loading, forcing it to accumulate in the approximate region of kinetochore microtubules. This localization suggests that SYS-1 may be directly transported to centrosomes along microtubules via the action of molecular motors. Given the minus-end directed movement implied by the localization at a microtubule organizing center, we focused on dynein and dynactin components as the primary minus-end directed motor. Preliminary data from microtubule-destabilizing and ATP-depleting chemical treatment indicate that both ATP and microtubules are important for SYS-1 centrosomal localization. A temperature sensitive allele of dynein heavy chain show a disproportionate effect on SYS-1 localization. Additionally, an RNAi screen aimed at depleting these microtubules and microtubule transport proteins has identified several dynein light chain subunits that promote proper centrosomal localization of embryonic SYS-1/beta-catenin. Together, these results suggest an active role for the multimeric dynein/dynactin cargo binding complex in SYS-1/beta-catenin regulation and signaling status.