Seydoux GC et al. (1994) Worm Breeder's Gazette "pes-10 RNA and Protein Are Expressed Transiently in Each Somatic Lineage in the Pre-Gastrulation Embryo"

Seydoux GC, Fire A

[Worm Breeder's Gazette, 1994]

pes-10 RNA and protein are expressed transiently in each somatic lineage in the pre-gastrulation embryo Geraldine Seydoux and Andy Fire. Carnegie Instituhon, Baltimore MD 21210.

Wannissorn, Nattha et al. (2011) International Worm Meeting "RNAi screening in Caenorhabditis elegans Ras mutant identified blmp-1 as a putative Ras cooperating tumor suppressor gene."

Fraser, Andy, Wannissorn, Nattha

[International Worm Meeting, 2011]

The key to successful diagnosis and treatment of cancer is not only to identify the individual mutations that drive tumorigenesis but also to understand how multiple mutations cooperate. Activating mutations in the Ras oncogene are found in a high proportion of human tumors; however, other mutations are required for full-blown cancer and identifying these additional mutations is crucial. In the first part of my project, I tackled this challenge by using RNAi screens to identify genes whose losses enhance phenotypes associated with activating mutations in let-60, the C. elegans ras orthologue. Human orthologues of these genes might thus play a similar cooperative role in ras-driven tumors. I found that RNAi knockdown of the PRDM1 ortholog blmp-1 strongly enhanced the activated Ras vulval phenotype. Although PRDM1 regulates several critical developmental processes and is a tumor suppressor gene, it has not been previously described as a regulator of Ras signaling. To dissect the mechanism by which blmp-1 loss cooperates with activated let-60 mutations, I carried out a second RNAi screen for genes that suppress the blmp-1; let-60 enhanced phenotype. These genes may be required for the cooperation between blmp-1 and let-60. I identified the Paired-box transcription factor gene pax-3 as a strong suppressor of the enhanced activated Ras phenotype. The data presented here suggests that BLMP-1 may repress pax-3 transcription directly and that activated Ras leads to attenuation of this repression. My future works are directed towards testing this model. In summary, I have shown that loss of blmp-1 strongly cooperates with activating mutations in let-60 and that this cooperation requires the direct BLMP-1 target pax-3. We predict that similar cooperation will occur between mammalian Ras and PRDM1 in human cancer.

Wannissorn, Nattha et al. (2009) International Worm Meeting "Identification and Characterization of Modifiers of the Multivulva Phenotypes in Caenorhabditis elegans Ras Mutant."

Wannissorn, Nattha, Fraser, Andy G.

[International Worm Meeting, 2009]

While mutations in ras are found in a high percentage of human tumours, additional mutations are required for progression from transformation to full-blown oncogenesis. Identifying such cooperating mutations is key for understanding the progression in humans. In C. elegans, worms homozygous for activating mutations in the ras orthologue let-60 have multivulva phenotypes (Muv), with approximately 60 percent of let-60(n1046) animals being multivulval at 20 deg C. Therefore, we use this phenotype as the basis for a large-scale RNAi screen to identify either enhancers or suppressors of the let-60(n1046) Muv phenotype. Since ras and many cooperating signaling pathways are well conserved between worms and humans, RNAi knockdowns that decrease the percentage of Muv worms are likely to uncover orthologs of protooncogenes, whereas knockdowns that increase the percentage of Muv worms should uncover tumor suppressor gene orthologs. Therefore, we use RNAi screening in C. elegans to identify novel cancer associated genes in humans. Rather than carry out genome-scale screens, we focused on two sets of genes to test initially. The first is a set of ~250 chromatin remodeling factors. Many chromatin remodeling factors have been implicated roles in cancer including p105Rb, histone acetyl transferases, and NuRD complex components. In worms, the SynMuv genes repress the vulval fate and they encode nuclear proteins that function principally through chromatin remodeling. Thus chromatin remodeling factors are likely to be enriched in regulators of Ras signaling in both vulval induction and carcinogenesis. We present here our data from screening all 250 chromatin remodeling factors for enhancers or suppressors of the let-60(n1046) Muv phenotype. The second set of genes was chosen as orthologues of human genes whose mutation in cancers is being studied in depth by The Cancer Genome Atlas Project. This is a collaborative effort to sequence tumor genomes from tumor samples. In the pilot projects, 623 known cancer-associated genes were selected for gene sequencing and we are screening all orthologues of these genes in the worm for enhancers or suppressors of the let-60(n1046) Muv phenotype. Since the cancer genomes may accumulate higher background mutation rates, the presence or the frequency of mutations uncovered by DNA sequencing may not indicate the biological relevance of the mutations themselves. We hope that the studies we are carrying out in the worm will aid assigning biological relevance to the identified mutations in tumours. We will present the results from these screens here.

Lautens, Margot et al. (2017) International Worm Meeting "The search for novel anthelmintic targets: Characterizing alternative metabolic pathways in Caenorhabditis elegans."

Caudy, Amy, Del Borrello, Samantha, Fraser, Andy G., Lautens, Margot

[International Worm Meeting, 2017]

Around a quarter of the human population is infected by parasitic helminths and they place a large economic burden on the agricultural industry. Unfortunately anthelmintic resistance is a growing problem. Although helminths are able to survive long periods of time of hypoxia in their hosts, their anaerobic metabolism has yet to be fully characterized. These alternative pathways are a promising, selective target for new drugs. The Fraser lab has developed a movement assay that allows for quantitive response to drugs and genetic perturbations to be measured in Caenorhabditis elegans, a free living relative who can also survive hypoxia. When the electron transport chain (ETC) is blocked by high doses of cyanide (KCN), worms are unable to move. However, if glycolysis is blocked at the same time by 2-deoxyglucose (2DG), the worms are able to recover. This two drug combination recapitulates the hypoxia response on a transcriptional level and this recovery requires the key hypoxia transcription factor, HIF-1. Furthermore, Complex I, an established hallmark of anaerobic metabolism and a target of existing anthelmintics, has been shown to be essential for this recovery. Together this strongly suggests that C. elegans recovers from KCN-induced paralysis in the presence of 2DG using anaerobic metabolism. Steady state LCMS has already revealed that the addition of 2DG allows for a buildup of succinate in worms treated with KCN alone to be depleted while the glycolytic shunt and the propionate shunts have been found to be nonessential for recovery. Two metabolic mutants in opposing pathways in the rate-limiting step TCA cycle show hyper- and hypo-recovery (idhg-2 and idh-2 respectively) from KCN in the presence of 2DG. These findings show that this system will allow us to find inhibitors of anaerobic metabolism and so, hopefully anthelmintic targets.

Braukmann F et al. (2017) RNA Biol "Artificial and natural RNA interactions between bacteria and C. elegans."

Jordan D, Miska E, Braukmann F

[RNA Biol, 2017]

19years after Lisa Timmons and Andy Fire first described RNA transfer from bacteria to C. elegans in an experimental setting [Timmons and Fire, 1998 ] the biological role of this trans-kingdom RNA-based communication remains unknown. Here we summarize our current understanding on the mechanism and potential role of such social RNA.

Wendy Wong et al. (2006) European Worm Meeting "The Integrative Interaction Map for Caenorhabditis elegans"

Wendy Wong, Andrew Fraser

[European Worm Meeting, 2006]

. Wendy Wong and Andrew Fraser. C. elegans is a popular model system for the systematic analysis. However, relatively little is known for its networks of genetic interactions. We hereby construct an integrative interaction map for C. elegans by integrating diverse functional genomics Datasets. We will also present some of the novel tools in querying the interaction map for answering powerful biological hypotheses.

Papp, Andy et al. (2015) International Worm Meeting "Low-cost LED-based Fluorescence Dissecting Stereomicroscope for multiple Fluorophores."

Biondo, John, Papp, Andy

[International Worm Meeting, 2015]

In previous studies (Papp et al., 2009, Papp et al. 2011) we demonstrated that high-power Light Emitting Diodes (LEDs) could be combined with good quality filters to visualize the expression of fluorescent transgenes and produce a low-cost fluorescent dissecting stereomicroscope system. Two of the cost saving methods were: (1) the use of oblique rather than epi- illumination and (2) putting emission filters into the eyepieces and/or camera port rather than using filter cubes with dichroic mirrors. While this system was shown to work well for multiple fluorophores (eGFP, dsRed, and mCherry) individually, it took about 1 minute to change lamps and eyepieces, making it impractical to screen individual moving worms for multiple fluorophores expressed in the same animal.In the present study, we investigate the use of multiple LEDs and filters in various lamp assemblies, and filter sliders, to reduce the switchover time between screening for two or more fluorophores. Preliminary data suggest that the switching can be done in under 3 seconds (a similar time scale to switching filter cubes in a conventional epifluorescence system) without a significant increase in cost or complexity as compared with the original low-cost system.Papp, Andy et al. (2009) International Worm Meeting "Investigation of Low-cost GFP Microscopy."Papp, Andy et al. (2011) International Worm Meeting "Investigation of Low-cost Fluorescence Microscopy."Papp, Andy et al. (2013) International Worm Meeting "Investigation of Simplified Dual-fluorophore Dissecting Stereomicroscopes.".

Way JC et al. (1988) Worm Breeder's Gazette "localization of mec-3 expression using mec-3 - lacZ fusions, and functional organization of mec-3."

Chalfie M, Way JC

[Worm Breeder's Gazette, 1988]

mec-3 is a homeobox-containing gene that is required for expression of differentiated characteristics of the touch receptor neurons (Way and Chalfie, Cell 54, 5-16). To identify the cells in which mec-3 is expressed, we constructed a mec-3-lacZ gene fusion by inserting a lacZ XhoI-SalI fragment into the XhoI site in the mec-3 homeobox, leaving 6 kb of upstream C. elegans sequence. This construction was injected into C. elegans along with Andy Fire's 'twitcher' plasmid (pD10.41, which transcribes the antisense strand of unc-22 from the unc-54 promoter). We identified one injectant from which the two plasmids formed a large, heritable extrachromosomal tandem array (termed uEx4). Animals carrying uEx4 can be recognized as twitchers. When animals are stained with X-gal according to Andy Fire's procedure, the touch receptors (ALMs, PLMs, AVM and PVM) are stained, as predicted. In any given animal, the complete set of cells is usually not stained, suggesting that these animals are genetic mosaics.

Lautens, Margot et al. (2021) International Worm Meeting "The Search for Novel Anthelmintic Targets: Characterizing Alternative Metabolic Pathways in Caenorhabditis elegans"

Serrat, Xenia, Fraser, Andy, Lautens, Margot, Schertzberg, Michael, Del Borrello, Samantha, Tan, June

[International Worm Meeting, 2021]

Around a quarter of the human population is infected by parasitic helminths and this places a large economic burden on the agricultural industry. Unfortunately anthelmintic resistance is a growing problem. Helminths survive long periods of hypoxia in their hosts, but their anaerobic metabolism has yet to be fully characterized. These anaerobic metabolic pathways are a promising, selective target for new drugs. Fumarate reduction, a rewiring of the TCA cycle, has long been identified as a key pathway for parasitic worms. We have previously identified the metabolic pathway for the synthesis of the essential small molecule involved in fumarate respiration, rhodoquinone (RQ) which combines two important pathways, the kynurenine metabolism pathway and the ubiquinone synthesis pathway and found that COQ-2 is the key connection between both. We have previously found that a mutually exclusive exon in coq-2 which is exclusive to RQ-species is what determines whether RQ or the closely related ubiquinone (UQ) is synthesized. coq-2 has two key residues near the active site which are conserved in RQ-species but not in other invertebrates or in hosts. Furthermore, mutating these two residues is sufficient to change the UQ-exon so that it can recover in a RQ assay developed by our lab. This finding combined with previous research which suggested that parasitic worms rely on extensive rewiring of existing pathways led to a focus on known metabolites and known pathways which often have existing drugs and crystal structures. By building from previous work, drug targets can be more easily found than if a novel pathway had to be characterized. The most effective drug targets will be those genes which are funadamental to parasitic metabolism and which have had divergence between hosts and parasites so that they can be targeted selectively. More specifically, four pathways which have been previously linked with anaerobic metabolism were tested for importance in RQ-dependent metabolism to narrow down to specific targets. For each of these targets, protein sequences were compared between hosts, parasites and RQ-containing mollusks and annelids to find conserved divergent residues in proximity to druggable sites. In total four known enzymes, including COQ2, have been found which are required for RQ-dependent metabolism and which have parasite specific residues near active sites which may act as selective drug targets. Humanized mutants have been generated for those residues. Moving forwards, in silico drug design will be carried out to find drugs predicted to be specific to the helminths which can be then be assayed against wild-type as well as the humanized mutants.

Papp, Andy et al. (2013) International Worm Meeting "Investigation of Simplified Dual-fluorophore Dissecting Stereomicroscopes."

Aldrich, Chris, Papp, Andy

[International Worm Meeting, 2013]

In previous studies (Papp et al., 2009, Papp et al. 2011) we demonstrated that high-power Light Emitting Diodes (LEDs) could be combined with good quality filters to visualize the expression of fluorescent transgenes and produce a low-cost fluorescent dissecting stereomicroscope system. Two of the cost saving methods were: (1) the use of oblique rather than epi- illumination and (2) putting emission filters into the eyepieces and/or camera port rather than using filter cubes with dichroic mirrors. While this system was shown to work well for multiple fluorophores (eGFP, dsRed, and mCherry), it took about 1 minute to change lamps and eyepieces, making it impractical to screen individual moving worms for two fluorophores expressed in the same location (we were able to view eGFP and dsRed simultaneously using a single excitation and emission filter set, but one is likely to obscure the other if they are co-localized). In the present study, we investigate the use of multiple LEDs and filters in the same lamp assembly and filter sliders to reduce the switchover time between screening for two fluorophores. Preliminary data suggest that the switching can be done in under 3 seconds (a similar time scale to switching filter cubes in a conventional epifluorescence system) without a significant increase in cost or complexity as compared with the original low-cost system. Papp, Andy et al. (2009) International Worm Meeting "Investigation of Low-cost GFP Microscopy." Papp, Andy et al. (2011) International Worm Meeting "Investigation of Low-cost Fluorescence Microscopy.".