Heather Thieringer et al. (2003) International Worm Meeting "Using C. elegans in a laboratory course designed for non-science majors."

Bonnie Bassler, Eric Wieschaus, Heather Thieringer

[International Worm Meeting, 2003]

MOL 101: From DNA to Human Complexity is an introductory undergraduate science course with a laboratory component designed for the non-science major. The students taking this course range from freshman to seniors. This lecture and laboratory course acquaints students with the theory and practice of modern molecular biology, with a focus on biological topics of current public interest. Within the laboratory component of MOL101, we have developed a two-week laboratory exercise that uses C. elegans to illustrate the concepts of reverse genetics and the use of model organisms to understand and investigate human disease. The C. elegans lab follows directly after a two-week lab that examines Drosophila embryonic mutations using Drosophila embryo cuticle preps to illustrate the concepts of forward genetics and the genetic control of development. These two labs work well together and allow the student to compare development and basic biological functions in two separate organisms.We have developed a protocol that enables the students to work with C. elegans without requiring the manipulation of nematodes with a worm pick. Prep work for the lab includes an egg prep with subsequent hatching on unseeded plates. Students are then able to add L1 starvation-arrested worms to the bacterial RNAi plates after washing with M9 buffer. In this way, one large egg prep can be made and used for multiple lab sections. The students are given 5 different bacterial cultures and a brief description of the genes that are being targeted by RNA interference. We chose a variety of phenotypes, including an unc, a bag-of-worms, a larval arrest, and an embryonic lethal phenotype. We are able to time the experiments so that students can observe the worms exactly one week after setting up the experiments. In the second laboratory section, students are provided with a checklist of characteristics and behaviors with which to examine each plate. Through observation of the resulting RNAi phenotypes they are asked to make predictions about the identity of each gene, and to further relate the gene and phenotype to the study of human biology and disease.

Irene Zelepuhin et al. (2005) International Worm Meeting "Genetics of sex Reversal of C. briggsae::C. remanei XO hybrids."

Scott Baird, Shannon Romer, Irene Zelepuhin

[International Worm Meeting, 2005]

Sterile F1 adult hybrids can be obtained from crosses of C. briggsae males to C. remanei females. Depending on the strains used, all hybrids are females. The absence of male hybrids results from sexual transformation, not male-specific lethality, as both XX and XO females are observed. Variant strains of C. briggsae and C. remanei suppress this hybrid sexual transformation, demonstrating genetic variation within both species the affects sex determination in F1 hybrids. This variation has been used to map hybrid sex reversal loci. In C. remanei, an ill-defined region of the X chromosome is implicated. In C. briggsae, three loci linked to hybrid sex reversal have been identified. The strongest linkage is to allelic variants in Cb-tra-2. A null mutation in Cb-tra-2 (thanks Eric) completely suppresses the transformation of XO hybrids. Other C. briggsae loci linked to sex reversal variants include Cb-daf-5 and Cb-mab-9.

Sawh, Ahilya et al. (2015) International Worm Meeting "Regulation of DCR-1-dependent RNAi pathways through DCR-1 phosphorylation."

Duchaine, Thomas, Wohlschlegel, James, Lewis, Alexandra, Sawh, Ahilya

[International Worm Meeting, 2015]

RNA interference (RNAi) pathways are directed by small (18-22nt) non-coding RNAs and orchestrate a variety of evolutionarily-conserved gene-silencing programs. The type III RNase protein Dicer is at the center of most of these pathways, including responses triggered by exogenous sources of long double-stranded RNA (exoRNAi). Other pathways, such as microRNA-mediated silencing and endoRNAi are triggered by endogenous loci, and control developmental and homeotic events. Dicer's role is crucial and three-fold: it cleaves long dsRNA precursors into smaller RNAs that direct silencing events, it loads the small RNAs into Argonaute proteins to form an active RNA-induced silencing complex (RISC), and it acts as a scaffold for several protein co-factors required for substrate recognition and processing. In C. elegans, Dicer (DCR-1) is essential in three small-RNA mediated silencing mechanisms including exoRNAi and ERI endoRNAi, two RNAi pathways that compete for downstream effectors. DCR-1 is part of distinct multi-protein complexes in each of these RNAi pathways: RDE complex in exoRNAi and ERIC in ERI endoRNAi.We discovered and validated several phosphorylated determinants of DCR-1 in the C. elegans. Particular attention was placed on a cluster of phosphorylated sites between the PAZ and RNase III domains, one of which could implicate AMPK signaling. Recent crystal structures of human Dicer have shown that this region could be involved in recognition of dsRNA substrates. We thus hypothesized that phosphorylation in this cluster could impair substrate recognition, or alter its specificity.To study the effect of DCR-1 phosphorylation on the three DCR-1-dependent RNAi pathways in C. elegans, transgenic strains were engineered expressing phosphorylation-deficient, and phosphorylation-mimetic mutants of DCR-1 in this cluster of sites. Functional analysis on these strains revealed that phosphorylation-deficient mutants display an impaired exoRNAi response, and severe developmental defects. Phosphorylation-deficient DCR-1 mutants also enhance interaction with ERI-endoRNAi DCR-1 cofactors. Taken together, these results support a model wherein DCR-1 phosphorylation may promote its shift from the ERIC to the RDE complex. Post-translational regulation of Dicer and RNAi pathways are largely unexplored and could potentially be incredibly impactful in light of their crucial roles in development and disease. The present study will shed light on how RNAi pathways integrate signals through phosphorylation signaling cascades.

Lee, Inhwan et al. (2013) International Worm Meeting "Epigentic regulation of L1 longevity."

You, Young-jai, Lee, Inhwan

[International Worm Meeting, 2013]

Animals encounter food infrequently and starvation is a common physiological state in their natural circumstance [1]. Hatching in the absence of food, worms arrest their development at the L1 stage and survive starvation more than two weeks [2]. It was shown that adult longevity is regulated epigenetically by chromatin alterations and the genes that regulate epigenetics [3]. Here, we investigate whether L1 longevity is also regulated epigenetically. When we measured various histone modifications using Western blot, we found histone 3 lysine 4 tri-methylation, known to be a modification to activate gene transcription, is increased in L1 starvation. Moreover, mutants of set-2, which encodes a histone 3 lysine 4 tri-methyl transferase that functions in adult longevity [3], has reduced L1 longevity. There are several genes essential for normal L1 longevity, such as aak-2 and daf-16. Based on our data, we hypothesize that chromatin remodeling through histone 3 lysine 4 tri-methylation regulates transcription of genes involved in L1 longevity. Currently we are testing this hypothesis by measuring expression levels these genes by ChIP-qPCR during L1 starvation in set-2 mutant. References [1] Brian H. Lee, Plus Genetics, 2008 [2] Inhwan Lee, Plus One, 2012 [3] Eric L. Greer, Nature, 2010.

Hamill, Danielle R. et al. (2009) International Worm Meeting "Analysis of a spindle assembly mutant in C. elegans."

Hamill, Danielle R., Everett, Molly, Bowerman, Bruce, Price, Meredith, McNeeley, Marie, Mazhar, Sahar, Gearica, Amy

[International Worm Meeting, 2009]

Cell division is a highly conserved and complex process that must be carefully orchestrated. or452ts was isolated in a screen for temperature sensitive embryonic lethal cell division mutants. In or452ts mutant embryos, bipolar spindles often fail to form. In these mutants DNA localizes around microtubules emanating from a single, often misshapen, centrosome. Staining with centriole markers suggests that a centriole duplication defect may underlie the spindle assembly defect. Using live cell imaging and fluorescence transgenes (including GFP::gamma tubulin), we have noticed centrosome streaking and splitting that is not seen in wild-type embryos. We also have observed the formation of asymmetric spindles. Towards the goal of identifying the gene that is mutated in or452ts, we have done meiotic mapping using phenotypic markers and snip-SNPs. We have narrowed the genetic location down to about 0.5 cM around +2.5 on LGIII. This region contains less than 500 kbp of DNA, and there are about 40 predicted genes in this region. In collaboration with Doug Turnbull and Eric Johnson (University of Oregon), we are sequencing the DNA from this region to try to identify the mutation responsible for the or452ts mutant phenotype. We believe that characterization of this mutant, and identification of the gene responsible, will provide new insights into the process of mitotic spindle assembly and cell division.

Knight JK et al. (1997) International C. elegans Meeting "gad-1 CONTROLS GASTRULATION INITIATION AND ENCODES A G BETA-LIKE PROTEIN"

Knight JK, Wood WB

[International C. elegans Meeting, 1997]

Gastrulation in C. elegans is normally initiated by the inward migration of the two gut precursor (E) cells at the 26-cell stage. We have isolated a gastrulation defective, temperature-sensitive mutation, ct226ts, defining a gene called gad-1 (gastrulation defective). ct226 exhibits a strict maternal effect, and appears to be a hypomorphic allele. At 25C, E cells divide early and in the wrong plane, and do not migrate into the embryo. Embryos continue to develop and differentiate all the major cell types, but do not undergo morphogenesis. We have rescued gad-1 homozygous mutant animals with a 4 kb piece of genomic DNA containing only one gene. The predicted translation product of this gene contains 6 WD-repeats most similar to those found in the beta-transducin family of G protein beta-subunits (1). From the comparatively low BLAST scores, gad-1 may encode a novel protein with a beta-subunit-like function. We sequenced the entire predicted coding region of gad-1(ct226), and found the molecular lesion in the third of five predicted exons. The mutation changes a His to an Asp in the most conserved WD-repeat in the predicted protein. Other maternal-effect genes with similar defective phenotypes to gad-1 have been identified (but not characterized), and embryonic transcription is also required for gastrulation initiation (2). These findings suggest that gastrulation initiation in C. elegans, as in Drosophila (3), may be mediated by a G protein signaling pathway, with gad-1 playing a novel regulatory role. 1. Neer et al. 1994, Nature 371, 297-300. 2. Powell-Coffman, Knight and Wood, 1996, Dev. Biol. 178, 472-483. 3. Parks and Wieschaus, 1991, Cell 64, 447-45558.

Megan A Higginbotham et al. (2003) International Worm Meeting "A mod-5 Suppression Screen for Genes Involved in Serotonergic Neurotransmission"

Megan A Higginbotham, Bob Horvitz

[International Worm Meeting, 2003]

Wild-type animals that have been acutely food deprived slow their locomotory rate upon encountering bacteria more than do well-fed animals. This behavior, called the enhanced slowing response, is serotonin (5-HT) dependent. Animals mutant for the 5-HT reuptake transporter mod-5 slow even more than wild-type animals. Additionally, mod-5 animals are hypersensitive to exogenous 5-HT. To identify additional genes involved in 5-HT signaling and possibly the enhanced slowing response, we screened for suppressors of this 5-HT hypersensitivity. We screened 46,200 haploid genomes using Mos1 transposon mutagenesis (Bessereau et al. Nature, 413: 70-74, 2001). Four strong suppressors were identified. Two contain insertions in genes previously known to suppress mod-5 for both the exogenous 5-HT hypersensitivity and the hyperenhanced slowing response. One of these suppressors is an allele of mod-1, which encodes a 5-HT-gated chloride channel, and the other is an allele of goa-1, a predicted alpha subunit of a heterotrimeric G-protein. Four transposon insertions have been identified on chromosome I in the strain carrying the third suppressor, n4094. Four transposon insertions have also been identified in the strain carrying the fourth suppressor, n4095. Experiments are underway to determine which of these insertions, if any, causes suppression of the 5-HT hypersensitivity of mod-5. We are also working to define neural circuits through which both mod-5 and suppressors of mod-5 act to affect the hyperenhanced slowing response. Using antibodies raised against MOD-5 protein, we have identified several head neurons in which MOD-5 expression can be seen. Additionally, a translational mod-1::rfp reporter has been constructed (by Eric Miska), and we are currently identifying the cells in which it is expressed.

Roussell DL et al. (1991) International C. elegans Meeting "A PUTATIVE RNA HELICASE FROM C. ELEGANS THAT SHARES HOMOLOGY WITH THE GERMLINE-SPECIFIC HELICASES, VASA AND PL10."

Roussell DL, Gharib S, Bennett KL

[International C. elegans Meeting, 1991]

We are interested in those genes (and their protein products) that establish the germline in the developing embryo of nematodes. Potential candidates for germline determinants include the protein component of the P-granules. Mutants that lack or improperly segregate these maternallysupplied granules do not develop a germline, are sterile, and are phenotypically grouped into a class of mutants appropriately called grandchildless (Schupbach and Wieschaus, Dev. Biol. 195:302-317; Martin et al., C. elegans Meeting Report, CSH, pp. 197). The first gene to be cloned that corresponds to a known grandchildless locus is the vasa gene from Drosophila melanogaster ( Lasko and Ashburner, Nature 335:611-617; Hay et al., Cell 55:577-587). We have cloned a potential vasa-equivalent from Caenorhabditis elegans by the polymerase chain reaction (PCR). Degenerate oligonucleotide primers were designed from the predicted vasa amino acid sequence. The PCR-amplified C. elegans DNA was purified, cloned and sequenced. Amino acid sequence comparisons show 48% perfect amino acid homology (and 66% homology with conserved amino acid changes) between the Drosophila vasa and the Caenorhabditis PCR clone that we call NCI. In addition, this clone shares 48% perfect amino acid homology with the mouse gene PL10, a male germ cell- specific helicase (Leroy et al., Cell 57:549-559). The NCI insert DNA hybridizes to a 6.4 Kb and 4.3 Kb DNA fragment on Southerns of EcoRI digested C. elegans genomic DNA. The NCI hybridization probe also identifies two overlapping YACs on the YAC grid (kindly provided by Bob Waterston), which places this putative RNA helicase gene on linkage group I between dpy-5 and unc-15. We do not yet know if this defines one or multiple genes. On Northern analyses of poly A+ RNA from mixed stage C. elegans, NCI hybridizes to a 2.6 Kb RNA and to a larger, relatively less-abundant RNA. The PCR clone, NCI, has also been used as a hybridization probe to isolate several C. elegans genomic clones from a lambda EMBL4 library that was previously constructed in this laboratory. Hybridization to one genomic clone, CEA-I, is confined to an EcoRI fragment of the same approximate size as that detected on genomic Southerns. We are also currently characterizing potential NCI cDNAs from a library provided by Stuart Kim. Sequence analysis of the genomic and cDNA clones should determine if the C. elegans clone is vasa-like, PL10-like, or another putative RNA helicase.

Kosalka, J. et al. (2015) International Worm Meeting "Mechanism and regulation of piRNA biogenesis in the C. elegans germline."

Weick, EM., Miska, E., Kosalka, J.

[International Worm Meeting, 2015]

Mechanism and regulation of piRNA biogenesis in the C. elegans germlineJoanna Kosalka, Eva-Maria Weick, Eric A MiskaWellcome Trust/CRUK Gurdon Institute/Department of Genetics, University of Cambridge, Cambridge, United KingdomPiwi-interacting RNAs (piRNAs) are the most recently identified and by far the largest class of endogenous small regulatory RNAs (sRNAs). In C. elegans, piRNAs are synthesised as single strand precursors from specific genomic loci known as piRNA clusters, processed into mature 21 nucleotides long, 5'U sRNAs that are recognised by the PIWI clade of Argonaute proteins. piRNAs are germline-expressed and are essential for fertility and genome stability. piRNAs can align to transposons and repetitive sequences in an antisense orientation, targeting them for silencing and the loss of the piRNA pathway results in reactivation of transposons. In addition, some endogenous protein-coding genes are under control of the piRNA pathway.Many aspects of the C. elegans piRNA pathway remain poorly understood and elucidating the biogenesis of 21U RNAs is of particular interest. Importantly, the fundamental questions of how the synthesis of piRNA precursors is initiated and directed to specific loci, and how the maturation of the precursor molecules is executed, remain unanswered. Furthermore, the developmental regulation of piRNA expression is unknown.In a forward genetic screen our lab has identified prde-1 (piRNA defective) as an essential germline-expressed factor for piRNA biogenesis (Weick et al. 2014). prde-1 is involved in the initial steps of piRNA biogenesis and is indispensable for piRNA precursor synthesis and/or stability. Interestingly, PRDE-1 protein localises specifically to parts of chromosome IV coinciding with the large piRNA cluster. Although the molecular mechanisms of prde-1 function are currently unknown, these findings provide an exciting starting point to elucidate the mechanisms of piRNA cluster transcription and regulation.Here we will present our most recent progress on understanding the molecular machinery required for the biogenesis of piRNAs. We used a range of biochemical approaches to identify and characterise PRDE-1-interacting proteins and their role in piRNA biogenesis.

Pilgrim, Dave et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "THE SEX DETERMINING PATHWAY IN CAENORHABDITIS; TRA SUPPRESSORS AND FEM GENES"

Reidy, Keith, Pilgrim, Dave, Egydio de Carvalho, Carlos, Dewar, Jill

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Sex determination in Caenorhabditis is a rapidly evolving process that can provide insights into how biological pathways can be modified from a common set of ancestral genes. C. elegans and C. briggsae are two of three androdiocious species within the Elegans group. These two species are both morphologically and developmentally similar but their genetic control of hermaphroditism is different. XX animals in these species are somatically female but are capable of producing and storing sperm before switching to oocyte production. Previous work from our lab and Eric Haag's showed that the function of the Tra genes was similar between the two species, while mutations in C. briggsae fem-2 or fem-3 genes resulted in somatic but not germline feminization, suggesting that the sperm/oocyte switch must be controlled downstream of the Fem genes in C. briggsae XX animals. We have now extended that work, by isolating and characterizing suppressors of Cb-tra-2 and Cb-tra-3 ts mutations, in hope of identifying missense mutations which will help with the analysis of Fem protein function.Over 70 tra-2 suppressors were identified in the Pilgrim and Haag labs and several tra-3 suppressors in our lab by forward genetics (looking for the restoration of self-fertility to tra(ts) animals at the restrictive temperature). The results of these screens and the ongoing characterization will be presented. The majority of the tra-2 suppressors are alleles of the three fem genes, as expected, including a large set of mutations in Cb-fem-1, which had not been previously isolated by reverse genetics. A small set of interesting mutations complemented the fem genes and are phenotypically distinct from any other mutants described in C. elegans or C. briggsae. WGS has identified candidates for these suppressors and we are currently in the process of mapping the mutations to confirm that the lesion is responsible for their unique phenotypes, and the alleles are being separated from the tra-2 background to allow characterization of their phenotype.