Lamunyon, Craig W. et al. (2011) International Worm Meeting "Y47D7A.16: a riboflavin tranporter in C. elegans and homolog of human Riboflavin Transporter 2."

Rothman, Jason, Biswas, Arundhati, LaMunyon, Craig W., Said, Hamid

[International Worm Meeting, 2011]

The C. elegans gene Y47D7A.16 is an ortholog of isoform 1 of the human Riboflavin Transporter 2 vitamin transporter. We became interested in this gene as the riboflavin transporter in C. elegans. Cloning of the cDNA revealed an exonic structure different from that predicted and an alternative splice in the first exon. Expression of the cDNA in human ARPE19 retinal cells showed enhanced riboflavin uptake, establishing the protein as a functional transporter of riboflavin (cRFT). Uptake by cRFT was pH dependent (uptake at buffer pH 5 > than uptake at pH 8), Na independent, and was inhibited by riboflavin structural analogues lumichrome and lumiflavin. Worms exposed to Y47D7A.16 RNAi laid defective eggs at a slowed pace and exhibited a slower defecation rate compared to controls. The protein is expressed primarily in the gut, with expression most pronounced in the distal gut, although expression also occurs in the excretory canal. We will discuss the effects of riboflavin supplementation on RNAi knockdown worms and on the expression of Y47D7A.16. [Supported by grants from the DVA and the NIH (DK-58057)].

Rothman, Jason et al. (2009) International Worm Meeting "Vitamin metabolism in a knockout mutant of the folate tranporter folt-1."

Craig, LaMunyon, Said, Hamid, Liau, Wei-Siang, Rothman, Jason, Ashokkumar, Balasubramaniem

[International Worm Meeting, 2009]

The C. elegans gene folt-1 is an ortholog of the human reduced folate carrier gene. The FOLT-1 protein has been shown to transport folate and to be involved in uptake of exogenous folate by worms. A knockout mutation of the gene, folt-1(ok1460) causes sterility due to defective oogenesis. Germ cell proliferation is drastically reduced, and knockout hermaphrodites contain few, if any, mature oocytes. On occasion, they also harbor several fertilized eggs containing defective embryos. Rarely, knockout hermaphrodites produce a small number of viable progeny with a brood size numbering less than 10. The knockout phenotype appears oogenesis specific because knockout worms produce functional sperm and knockout males are able to sire cross progeny. The lifespan of folt-1 hermaphrodites is severely diminished, suggesting that FOLT-1 functions in the soma as well as in the germline. Here, we investigate the effects of vitamin supplementation and vitamin uptake in folt-1 knockout worms. For thiamine, knockout worms upregulate thiamine uptake, and thiamine supplementation improves germline proliferation but does not correct the defects of the folt-1 knockout on embryonic development. We will describe similar investigations of several vitamins and argue that C. elegans vitamin metabolism is responsive to dietary availability.

Daniel P Blanchard et al. (2004) West Coast Worm Meeting "Context dependant interactions in RNAi"

Jamie Fleenor, Andrew Fire, Susan N Parish, Daniel P Blanchard

[West Coast Worm Meeting, 2004]

RNAi is a highly conserved sequence specific gene silencing mechanism where the intracellular presence of dsRNA causes the degradation of cognate mRNAs. Beside its ever increasing use as a selective gene repressor in reverse genetics and potential therapeutic uses, processes related to RNAi seem to be at the center of a number of endogenous gene regulatory mechanisms. Depending on the interaction between the trigger and target molecules, small RNAs can lead to a variety of outcomes, including mRNA degradation, translational repression, transgene silencing and epigenetic modifications. In order understand how protein interactions influence pathway selection we have developed an in-vivo binding assay based on differential cytolocalization of GFP tagged chimeras, which allows us to follow protein-protein interactions in the cytoplasm of metazoan cells. Utilizing this method we have assayed a subset of proteins directly or peripherally involved in RNAi confirming a number of previously documented interactions, thus validating the assay. Several of the proteins involved in RNAi readily interact with each other, presumably forming multimeric complexes which seem to be self sustaining rather than dependant on a particular scaffold protein, as determined by mutant analysis. Expression levels of proteins involved in RNAi seem to vary in a context dependant fashion. Moreover, protein levels seem to be modulated by the presence of other RNAi involved proteins. Through mutational analysis we are trying to determine which domains are responsible for individual interactions and the effect of abrogating the said interactions.

Toch, Katarzyna et al. (2021) International Worm Meeting "Sign and reciprocal sign epistasis across different environments"

Labocha, Marta, Buczek, Mateusz, Toch, Katarzyna

[International Worm Meeting, 2021]

Epistasis is a phenomenon of different loci interacting with each other, ergo having non-independent effect. But even though genetic interactions are widely spread across genomes their importance is still being questioned. Depending on the perspective, epistasis is said to either play an essential role in both applied (animals and plants breeding, forensics, personalized medicine) and evolutionary biology (emergence of sex, speciation, shaping evolutionary landscapes) or to be seen as a noise of linear genes effect. In general, epistasis can be either positive, when double mutant has higher than expected fitness, or negative, when the fitness is lower than expected. Sign and reciprocal sign epistasis are peculiar cases of genetic interactions, where one or both mutations have effect opposite from both mutations combined. We have tested over 1000 pairwise interactions in standard conditions and in three different environmental stressors: heat shock (37°C), oxidative (H2O2) and mutagenic (MMS) stress. We have found that sign epistasis is a prevalent event; 40-78% of all significant interactions are in fact sign epistasis. What is even more interesting, we were able to detect few examples of reciprocal sign epistasis, however only in harsh environments; one in MMS treatment and four in H2O2 treatment. All of them were the examples of positive reciprocal sign epistasis - despite the deleterious effect of both single mutations, double mutant exhibited higher than expected fitness (higher than animals without any mutations). Both sign and reciprocal sign epistasis are a game changer when it comes to predicting fitness landscapes. As much as sign epistasis is able to slow down the process of reaching fitness optimum, reciprocal sign epistasis can actually block it, unless e.g. two mutations occur simultaneously. Our findings are notably important, since they point to the importance of considering environment in studying genetics and evolutionary scenarios.

Bhatla, Nikhil et al. (2009) International Worm Meeting "Investigating C. elegans Learning as a Possible Approach to the Study of Consciousness."

Horvitz, Bob, Bhatla, Nikhil

[International Worm Meeting, 2009]

To study the mechanisms of consciousness, it would be useful to identify a highly manipulable organism that has perception, a key element of consciousness. I define perception to mean the ability to have an inner, qualitative experience of one''s environment, beyond simply sensing and responding. Perception is the subjective experience of what something feels like, such as sugar tasting sweet, snow feeling cold, and 700 nm light looking red. One way to determine whether an organism is capable of perception is to determine whether it can be aware of its surroundings. In people, awareness can be measured indirectly by a trace conditioning experiment. In this experiment, a neutral stimulus is presented and removed, and after an appropriate period of time a different, aversive stimulus is presented and removed. Each trial is repeated many times. A person is said to have learned if she starts responding to the neutral stimulus in an aversive way. On average, those who successfully learn are aware, meaning that they verbally report that the neutral stimulus (e.g. sound) predicts the aversive stimulus (e.g. an air puff to the eye or an electric shock). Note that if there is no temporal delay between the stimuli, learning no longer correlates with awareness (Clark & Squire, Science 1998; Carter et al., PNAS 2003). Since trace conditioning correlates with awareness, it might be possible to use this type of learning to test whether non-verbal organisms have the ability to perceive. I plan to investigate whether C. elegans can perform trace conditioning. Specifically, I plan to pair a neutral odor with carbon dioxide, which causes locomotion reversals (Hallem & Sternberg, PNAS 2008). If the presentation of the neutral odor alone is sufficient to induce reversals after the training period, I plan to screen for worms that cannot perform this task. In this way, I hope to identify genes and cells required for trace conditioning in worms. Perhaps these same genes will also be involved in perception and consciousness in worms and other animals.

Cohen, Robert D. et al. (2013) International Worm Meeting "A Functional Genomic Screen for the Telomerase RNA in C. elegans."

Smith, Christopher, Cohen, Robert D., Chu, Diana

[International Worm Meeting, 2013]

Telomerase maintains telomere length in eukaryotes by synthesizing telomere repeat patterns on the chromosome ends. It is composed of a protein component (TERT) with reverse transcriptase activity, and an RNA component (TERC) that acts as a template for the reverse transcriptase domain. As dysfunction in telomere length maintenance has been implicated in cancer and aging, the study of telomerase has emerged as an attractive research area for therapeutic intervention. Although identification of TERT genes in newly sequenced genomes has been achieved by homologous search with known TERT genes, the same cannot be said for TERC genes, as primary sequence is not well conserved. TERC identification has relied on co-purification experiments; however, these methods are costly and labor intensive, and as a result, TERC discovery has been limited. To identify candidate TERC genes in a set of genomes from closely related species, we have developed a novel bioinformatic strategy. C. elegans is an ideal organism for the technique, because its TERC gene is unknown, a rich body of bioinformatic resources are available, and RNAi assays are available to test candidate gene function by reverse genetics. Although primary sequence is not well conserved in TERCs, one feature is dependably reproduced - the 1.5x template repeat pattern (eg, 9 bases of the telomere repeat: TTAGGCTTA), which serves as a template for TERT. Searching the C. elegans genome for all instances of this sequence yields 20503 loci. We used the Mauve genome aligner to identify syntenic regions between the Caenorhabditis genomes. 1.5x template repeats conserved at the same syntenic locus in each species are high probability TERC gene candidates. As a positive control, this bioinformatic approach analyzed five vertebrate genomes to identify the human TERC gene, which has been previously published. We have verified expression and RNAi knockdown of two TERC gene candidates, and are currently developing a C. elegans telomere length assay to confirm a reduced telomere length phenotype. Identification of the C. elegans TERC would provide an important tool to elucidate telomerase function in C. elegans.

Yasumi Ohshima et al. (2006) East Asia C. elegans Meeting "Food, feeding, digestion and defecation of the worm"

Yasumi Ohshima, Yusuke Kaku, Yuta Yoshidome, Yuji Osako, Katsuhiro Kunigo, Akiko Nakasone, Kohji Miyahara, Tomonori Nakamura

[East Asia C. elegans Meeting, 2006]

Food or nutrition is vital for the life of C. elegans as for other animals, and should affect growth, body size and activity of the worm. In the laboratory, worms usually feed on OP50, a uracil-requiring strain of E. coli, which is a good food for worms in the sense that it supports rapid growth and production of many progeny. However, E. coli is an enteric bacterium that may not be abundant in soil which is said to be the natural habitat of the worm. Avery & Shtonda (2003) identified from soil samples more than 10 bacterial species that supported growth of the worm. Yet, the entire image of the natural food of the worm is not clear. Although the mechanisms of feeding and defecation of E. coli have been extensively studied, much is still to be solved, for example, the mechanisms of food recognition, rate and target of defecation. Furthermore, virtually nothing is known on the mechanisms of digestion of food and absorption of nutrients in the gut of the worm. In mammals, many factors which seem to be involved in digestion or absorption are known, but there is little genetics in any animal. Thus, we have begun studies on some of these, and plan to ask further, as follows. 1) Heat-killed E. coli is not likely to support growth of C. elegans. 2) We are studying whether selected microorganisms are good food or not. For example, yeast S. cerevisiae probably does not support growth to adults. 3) We are trying to identify live or dead microorganisms in the gut of marked worms that were put into a soil sample, by DNA sequencing. 4) Several artificial microspheres of various size or chemical nature are used to get clues to food recognition, feeding and defecation. 5) We are trying to analyze the time course of feeding, digestion and defecation of GFP-marked E. coli. 6) We have found many C. elegans genes possibly involved in digestion or absorption based on amino acid sequence homology with those in mammals. We have obtained KO mutants for some of them from Dr. S. Mitani for reverse genetics. 7) We have begun isolation of mutants on digestion or absorption. The screening at present uses GFP-marked E. coli as food. Two candidate mutants were obtained that retain more GFP fluorescence in the gut without little change in the pumping cycle.

Bhatla N et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Investigating C. elegans Learning as a Possible Approach to the Study of Consciousness"

Horvitz B, Bhatla N

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

To study the mechanisms of consciousness, it would be useful to identify a highly manipulable organism that has perception, a key element of consciousness. Perception can be defined as the ability to have an inner qualitative experience of the environment, beyond simply sensing and responding. Perception is the subjective experience of what something feels like, such as sugar tasting sweet, snow feeling cold, and 700 nm light looking red. One way to determine whether an organism is capable of perception is to determine whether it can be aware of its surroundings. In people, awareness of one's environment can be measured indirectly by a learning paradigm called trace conditioning. In each trial of a trace conditioning experiment, a neutral stimulus (e.g. sound) is presented and removed, and after a period of time (e.g. 1 second) a different, aversive stimulus (e.g. an air puff to the eye) is presented and removed. After several trials, a person is said to have learned if she starts responding to the neutral stimulus in an aversive way. On average, those who successfully learn are aware of their environment, meaning that they verbally report that the neutral stimulus predicts the aversive stimulus. A slightly different version of this experimental design, called delay conditioning, occurs when the aversive stimulus is presented before the neutral stimulus is removed, and the two stimuli coterminate. Whether delay conditioning also correlates with awareness is still a matter of debate (Clark & Squire, Science 1998; Papka et al., Psychological Science 1997; Lovibond & Shanks, J. Exp. Psych. 2002). Since trace conditioning (and possibly delay conditioning) correlates with awareness, this type of learning might serve as a test of whether non-lingual organisms have the ability to perceive. I am investigating whether C. elegans can perform delay and trace conditioning. Specifically, I am conducting experiments pairing various odorants (e.g. isoamyl alcohol) with aversive stimuli (e.g. violet light, carbon dioxide). If the presentation of the odor alone is sufficient to induce an avoidance response after the training period, I plan to seek mutant worms that cannot perform this task. In this way, I hope to identify genes and neural circuits required to perform delay and trace conditioning in worms. Perhaps these same genes will also be involved in perception and consciousness in other animals.

Placentino, M. et al. (2017) International Worm Meeting "Dissecting 21U RNA biogenesis and activity in the germline and the early embryo."

Placentino, M., Butter, F., Rodrigues, R., Ketting, R., Dietz, S., Domingues, A.

[International Worm Meeting, 2017]

Many different small RNA pathways exist in C. elegans. Some of these affect the expression of 'regular' genes, while others are more geared towards the silencing of repetitive elements in the genome. In general, they have a large impact on fertility. Most of these pathways, with the notable exception of miRNAs, act in a two-step manner. First, so-called primary Argonaute proteins, such as PRG-1 that binds to 21U RNAs, recognize a target transcript based on homology between small RNA and target RNA. This is followed by a second step in which an RNA-dependent RNA polymerase (RdRP) enzyme is recruited to the target, resulting in 22G RNA production. As the target-RNA is used as template by the RdRP, 22G RNAs are anti-sense with respect to the target of the primary small RNA. 22G RNAs are bound by secondary Argonaute proteins that induce silencing. In case of 21U RNAs, the secondary phase can become self-sustainable and independent of PRG-1, in which case the target is said to be under control of RNAe (RNA induced epigenetic silencing). The molecular differences between a PRG-1 target that is under control of RNAe and one that is not are presently completely unclear. We are further dissecting both the biogenesis of 21U RNAs as well as how and when 21U RNAs induce RNAe. Based on IP-mass spec experiments on PID-1, a factor we previously showed to be essential for 21U RNA production, we found evidence for a complex that drives the production of 21U RNA. The identity of the PID-1 interactors has implications for 21U RNA precursor recognition and the stabilization and/or transfer of processing intermediates. We established that 21U RNA-mediated silencing most likely takes place during early embryogenesis, since maternally provided 21U RNAs are both required and sufficient to induce an RNAe-like state. We then identified a novel protein, PID-2, that is required for PRG-1-mediated silencing activity, but is dispensable for RNAe. In pid-2 mutants, PRG-1 is normally loaded with 21U RNAs. Therefore, PID-2 acts downstream of PRG-1 loading, but upstream of the secondary RNAe pathway that is PRG-1 independent. Consistent with PRG-1 silencing activity in the early embryo, zygotic PID-2 is sufficient to induce silencing. We are currently further dissecting PID-2 activity, but it is already clear from our analyses that PID-2 defines an as yet poorly understood step in between target recognition by PRG-1 and establishment of a stably inherited RNAe-state.

Herman MA et al. (1996) Midwest Worm Meeting "GENES THAT CONTROL CELL POLARITY IN C. ELEGANS"

Kari CK, Herman MA, Herman RK, Shaw JE

[Midwest Worm Meeting, 1996]

When a cell divides to generate daughters of different cell fates, the division is said to be asymmetric. In C. elegans, 807 of the 949 nongonadal cell divisions that occur during development are asymmetric. An important feature of most asymmetric cell divisions in C. elegans is that they occur with a defined orientation with respect to the body axes. The relative orientation of the different daughter cells to each other and to the body axes gives an asymmetric cell division a polarity. Mutations in lin-44 cause the polarities of certain asymmetric cell divisions in the tail to be frequently reversed, while maintaining the asymmetries and the orientations of division planes. Genetic analysis indicated that the polarity reversal defect is the result of a complete loss of lin-44 function (Herman and Horvitz, 1994, Development, 120: 1035-1047). Molecular analysis has revealed that lin-44 encodes a member of the Wnt family of secretory glycoproteins, which have been shown to function as short range signaling molecules. Examination of animals containing reporter constructs designed to mimic patterns of lin-44 expression, as well as in situ hybridization experiments, have demonstrated that lin-44 is expressed in the developing tail hypodermis, which is posterior to the cells affected by lin-44 mutation. Mosaic analysis has shown that lin-44 function is not required in the cells affected by lin-44 mutation, but in cells in which we observe expression. Based upon these results we propose that lin-44 protein is secreted by the tail hypodermal cells and affects the polarity of asymmetric cell divisions that occur more anteriorly in the tail (Herman et al. Cell, 83: 101-110). As a result of the reversal of T cell polarity in lin-44 mutants, the phasmid socket cells are misplaced, and dye filling by the phasmid neurons is blocked. To identify other genes that act with lin-44 to specify cell polarity, we are screening for additional mutations that reduce or abolish the dye filling of phasmid neurons without affecting amphid neuron dye-filling. We have isolated over 40 such mutations. Many of these have been scored for the presence of the phasmid socket cells in their normal positions. Two mutations lacked phasmid socket cells in the normal positions: one is a new lin-44 allele and the other, mn553, identifies a new gene, which maps to LGII between dpy-10 and unc-4 and appears to cause defects in gonad morphology as well as in T cell polarity. The map position and phenotype of mn553 suggest that it might be an allele of mig-5, which has been shown by Guo and Hedgecock (WBG v14 n1, p. 52) to be involved in cell fate determination and cell migration and to encode a homolog of the Drosophila dishevelled (dsh) gene. We thus suggest that mig-5 may also be involved in the control of cell polarity. We also expect to identify mutations in lin-17, which cause a loss of T cell polarity and hence block phasmid dye-filling. lin-17 has been shown by Sawa and Horvitz (1995 Meeting Abstracts, p. 455) to encode a homolog of the Drosophila frizzled (fz) gene. In Drosophila, fz , wingless (a member of the Wnt gene family) and dsh mutants have defects in tissue polarity. Many structures on the cuticular surface of Drosophila display a tissue polarity characterized by arrays of polarized sensory bristles or hairs. This polarity appears to be controlled by a cell signaling pathway involving fz (lin-17), dsh (mig-5) and wingless (lin-44). We are exploring the possibility that these genes may interact in a similar manner to control cell polarity in C. elegans.