Wong S et al. (1996) Midwest Worm Meeting "NUCLEAR HORMONE RECEPTORS: WHEN ARE THEY EXPRESSED IN THE DEVELOPING NEMATODE?"

Wright LM, Wong S, Sluder AE

[Midwest Worm Meeting, 1996]

Forty-two genes encoding nuclear hormone receptors (NHR) have been identified in C.elegans but, for the majority, knowledge of their biological functions is scant. For most of these NHRs, we have only gene sequences that identify them as members of this superfamily of transcription factors. Two genes, nhr-2 and nhr-20, have been shown previously to be expressed embryonically and in all stages respectively. We are surveying the temporal expression profiles for additional genes in order to obtain clues to their functions. Of specific interest for further study will be genes exhibiting temporally restricted expression patterns that may indicate regulation of individual developmental events. We plan also to focus on NHRs that are conserved in parasitic nematodes, as nematode-specific hormone receptors could provide effective targets for novel antihelminthic drugs. Profiles for nhr-4, nhr-6, and nhr-18 were obtained using reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot analysis is underway to confirm the RT-PCR results. Of particular interest, our RT-PCR data for nhr-4 indicates a peak of expression during the late larval stages, a time when many crucial events occur in preparation for the larva-to-adult molt. In concert, we are also conducting mutant screens using Tc1 transposon and chemical mutageneses. These efforts focus currently on nhr-4, due to its limited temporal expression, and nhr-6, for which a homolog has been identified in the filarial parasite dog heartworm (C. Maina, pers. comm.). Future work will include examination of the spatial expression patterns of these genes.

Wong, WR. S. et al. (2017) International Worm Meeting "Phenotypic profiling of missense variants implicated in autism spectrum disorders."

Kato, M., Brugman, K., Wong, WR. S., Sternberg, P. W., Gharib, S., Maher, S., Oh, JY.

[International Worm Meeting, 2017]

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a strong genetic component. Human genetic studies have identified thousands of ASD-associated variants in over 850 genes. Understanding the physiological consequences of these variants will help characterize the essential components of ASD-associated genes. Here, we use C. elegans as genetic model to prioritize autism candidate alleles from the Simons Foundation Autism Research Initiative. We established an analysis pipeline. First, we use bioinformatics to filter human ASD-associated missense variants conserved in C. elegans protein sequence. Second, we construct the missense mutants using CRISPR/Cas9 and homology-directed repair knock-in technique. To analyze potential causal effects of autism-associated alleles, we then compare the phenotype of autism-associated alleles to the wild-type and the known loss-of- function allele of the same gene. For example, Leu1220Pro and Arg2624Gln missense mutations in chd-7, worm orthologs of human CHD7/CHD8, displayed decreased brood size compared to wild-type. Val369Met and Lys371Ser mutants of egl-19, worm ortholog of human CACNA1D, displayed subtle locomotion changes revealed by a quantitative locomotor tracking system. Our approach will help characterize novel missense alleles with human disease relevance and filter functional important alleles. This information will allow interpretation of these variants in genetic studies, and offer clear priorities for detailed studies in vertebrate models or human cells.

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.

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "Transgenic C. elegans expressing the fungal prion protein HET-s as a model for the study of amyloid infectivity and toxicity."

Qiao, Yujie, Saupe, Sven, Pinan-Lucarre, Berangere, Driscoll, Monica

[International Worm Meeting, 2009]

Amyloids are protein fibers rich in beta sheet structure that are associated with numerous neurodegenerative diseases, including Alzheimer disease. Amyloids have two major biological properties. First, they can be infectious--meaning that they can spread the altered amyloid conformation to the same protein or even among proteins of distinct primary sequence (the latter phenomenon is known as cross-polymerization). Infectious amyloids are called prions. Second, amyloids can be toxic, whether they are infectious or not. Both properties have tremendous fundamental and biomedical impact. [Het-s] is a prion of the fungus Podospora anserina involved in a defense cell suicide mechanism named heterokaryon incompatibility, which disables mixing of different strains by somatic fusion, an ubiquitous feature in filamentous fungi. The HET-s protein exists in a soluble state or in an aggregated, prion state named [Het-s]. The [Het-s] prion is not toxic by itself in P. anserina or in yeast. However cell death is rapidly triggered by the lethal interaction between 2 alleles of the het-s locus: het-S (het-"large S") and het-s (het-"small s") when the HET-s protein adopts the prion form. This prion has been extensively characterized and it is the only prion for which tridimensional structure is available to date. The critical point is that in the [Het-s] system, transgenic expression of het-s allows propagating a non-toxic prion while co-expression of het-s and het-S generates toxicity. We constructed strains expressing het-s or the prion domain only het-s(218-289) (the 218-289 fragment of the protein) as GFP fusions under the control of the ubiquitous promoter dpy-30. The fluorescence of pdpy-30het-s::gfp appears to be mainly diffuse in the cytoplasm while it shows strong aggregation in pdpy-30het-s(218-289)::gfp strains. We constructed strains expressing het-s(218-289)::gfp in the touch neurons using mec-4 promoter and observed that the fusion protein is mostly aggregated into a large fiber spanning the cell body and the proximal part of the axon. These aggregates remain to be shown as infectious [Het-s] amyloids. In future experiments, we will aim to generate neuronal toxicity through the co-expression of het-s(218-289) and het-S. This C. elegans model for prion studies, filling a gap between simple fungal systems and highly complex mammalian systems, might allow a better understanding of the molecular and cellular basis of amyloid infectivity and toxicity.

Lemieux JY et al. (1997) International C. elegans Meeting "TOWARDS THE MOLECULAR CHARACTERIZATION OF THE BIOLOGICAL TIMING GENE gro-1"

Lakowski B, Barnes TM, Lemieux JY, Hekimi S

[International C. elegans Meeting, 1997]

In a screen for C. elegans viable maternal-effect mutants, a number of novel phenotypic classes were defined (1). One of these classes is defined by the three clk genes, mutations in which result in a mean lengthening of development and life span, and an increase in the periods of rhythmic behaviors such as defecation, swimming and pumping (1). We have found that a fourth gene, gro-1, also shows the Clk phenotype (2,3). gro-1 was first recovered by Jonathan Hodgkin as a spontaneous segregant from a wild strain (personal communication). Of the four genes, only clk-1 has been thoroughly studied; a comprehensive phenotypic analysis was carried out by Wong et. al. (2) and the molecular cloning by Ewbank et. al. (4). The molecular characterization of each of the Clk genes will aid in understanding the Clk phenotype and ultimately the processes in which these genes are involved. We have mapped gro-1 to the interval between dpy-17 and clk-1 on LGIII. Injection of individual cosmids spanning this region identified three overlapping cosmids which were capable of rescue. Using the available genomic sequence, we tested individual predicted genes from the common rescuing region. This led to the identification of a single gene necessary and sufficient for rescue. The subsequent sequencing of the gene from the gro-1 mutant strain identified the e2400 lesion, confirming the gene assignment. Studies are now underway to determine the expression pattern of GRO-1. Details will be presented at the meeting. 1. Hekimi, S. et. al. Genetics 141: 1351-1364. 2. Wong, A. et. al. Genetics 139: 1247-1259. 3. Lakowski, B. and S. Hekimi. Science. 272: 1010-1013. 4. Ewbank, J. J. et. al. Science 275: 980-983.

Kotera, I. et al. (2011) International Worm Meeting "In vivo calcium imaging of the pharyngeal muscles in freely moving animals."

Su, L., Suzuki, H., Kotera, I., Truong, K., Wong, S.

[International Worm Meeting, 2011]

Imaging Ca2+ concentrations in live cells provides temporospatial dynamics of cellular activities, which is of vital importance in understanding how multi-cellular systems function. The first in vivo Ca2+ imaging in C. elegans has been established in the pharynx using a genetically encoded Ca2+ probe, cameleon; the pharynx is an attractive model for its neuronal regulation because the pharyngeal nervous system has few neurons, and functions semi-autonomously. However, previously reported imaging methods require immobilization of the worms and exogenously added serotonin just to observe pharyngeal activity. We have developed a new imaging system, which combines two independent imaging units to enable two simultaneous imaging, along with "worm tracker" functionality to perform imaging on a freely moving worm. We have also developed a pattern matching algorithm, which recognizes the expression pattern of the probe, and automatically extracts Ca2+ dynamics of the pharynx. Pharyngeal Ca2+ dynamics of freely-moving worms was measured in the presence of serotonin. Both the rise time (0.14s) and firing rate (3.14 Hz) of the transients in the pharynx are in good agreement with the previous report, confirming the validity of our new system. The free worms showed pharyngeal activity even in the absence of exogenous serotonin, albeit a reduced firing rate (0.24 Hz). The rise slope of the transients stayed relatively unchanged with or without serotonin (106%/s and 65%/s), but the rise duration has increased from 0.14s with serotonin to 0.39s without it. With an incorporation of new GCaMP, our result has demonstrated over 10-fold improvement in both the temporal resolution and dynamic range when used on our system. By employing all the techniques developed, an intriguing relation was revealed between the pharyngeal firing rate and locomotion direction. When worms are moving forward, the firing rate was similar to what we have measured with the serotonin-stimulated worms. However, when the worms autonomously reversed its direction, the firings quickly ceased; this pharyngeal firing was restored when the worm resumed forward movement. Thus we have developed a novel imaging system which is capable of concurrently performing Ca2+ imaging and transmission image acquisition on a freely moving worm. The ability to record multiple behaviors of the worm provides a unique opportunity to investigate how multiple systems are interacted and coordinated; further investigation of the seemingly isolated pharyngeal and somatic nervous systems should yield a compelling model for the inter-systems relation.

Ewbank JJ et al. (1997) International C. elegans Meeting "Expression and activity of clk-1"

Ewbank JJ, Lemieux JY, Felkai S, Labbe J-C, Hekimi S

[International C. elegans Meeting, 1997]

Mutations in clk-1 affect biological timing and extend longevity (Wong et al., Genetics, 139: 1247, (1995); Lakowski and Hekimi, Science, 272: 1010, (1996)). clk-1 encodes a protein of 187 amino acids that is highly conserved from yeast to mammals. The yeast homologue is the metabolic regulator Cat5p/Coq7p. clk-1 complements the phenotype of cat5/coq7 mull mutants, demonstrating that clk-1 and its yeast homolgue have a common biochemical function (Ewbank et al., Science, 275: 982, (1997)). We have now found that clk-1 is expressed at high levels in all somatic cells throughout development. Furthermore, under heat shock conditions the transcription of clk-1 and/or the stability of its message requires the activity of CLK-1. We are currently using Northern analysis, reporter constructs and antisera to study clk-1 expression further. We are particularly interested in the effects of temperature on clk-1 expression and determining the subcellular localization of CLK-1. We are also analyzing the phenotypic effects of everexpressing wild type or mutant versions of CLK-1 and investigating the molecular correlates of the maternal effect displayed by clk-1 (Wong et al., (1995)).

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Oppenheimer AJ et al. (1995) International C. elegans Meeting "G-PROTEIN SIGNALING IN C. ELEGANS"

Kaplan JM, Oppenheimer AJ

[International C. elegans Meeting, 1995]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate behavioral phenotypes dependent on G-protein signaling, we targeted expression of mammalian G-proteins to a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Ectopic expression of several G-proteins under the control of the not-3 promoter results in behavioral defects. Expression of constitutively active rat G-alpha-i1 causes uncoordinated forward and backward locomotion. Expression of wild-type rat G-alpha-s impairs backward locomotion, while constitutively active rat G-alpha-s causes defective backward locomotion and lethargy. In order to identify components of the G-alpha-s signaling pathway, we have initiated a screen for mutations that suppress the behavioral effects of activated G-alpha-s expression. In a screen of approximately 7000 haploid genomes, we have identified 11 independent mutations that improve backward locomotion. Several of these mutations result in a hyperactive phenotype in the absence of activated G-alpha-s expression. Mapping and complementation tests will determine whether the suppressors are allelic to other hyperactive mutants isolated by D. Elkes and J. Kaplan. By cloning the suppressor genes, we hope to identify ion channels regulated by G-alpha-s as well as other components of the G-alpha-s signaling pathway. We also plan to determine whether the behavioral effects of G-alpha-s expression are mediated by known second messenger systems. We are first testing the role of cAMP-dependent protein kinase (PKA), because it is activated by G-alpha-s in other systems and has been shown to phosphorylate ion channels. If G-alpha-s signals through PKA, increasing the level of PKA activity should mimic the effect of activated G-alpha-s expression, and inhibiting PKA should suppress the effect of activated G- alpha-s expression.

Hipsher LW et al. (1998) East Coast Worm Meeting "nhr-6: A MEMBER OF THE NGFI-B CLASS OF ORPHAN NUCLEAR RECEPTORS"

Wong S, Sluder AE, Hipsher LW, Maina CV

[East Coast Worm Meeting, 1998]

The nuclear receptor superfamily comprises the largest known group of transcription factors, members of which are found exclusively in metazoans. nhr-6 is one of the 210 nuclear receptor genes present in C. elegans (see abstract by Lindblom, et.al.). The apparent homology of nhr-6 to the vertebrate nerve growth factor induced protein B (NGFI-B) family of orphan receptors identifies nhr-6 as one of the few C. elegans nuclear receptor genes that fall into NR classes conserved between vertebrates and invertebrates. This conservation implicates nhr-6 as an ancient member of the NR superfamily, indicating potentially conserved and important functions. Furthermore, the presence of an nhr-6 homologue in the parasitic dog heart worm, Dirofilaria immitis, suggests that information pertaining to the biology of D. immitis could be gained via studies of nhr-6 function. In order to gain initial insight into the function of the nhr-6 gene product, we have utilized RNAi to disrupt gene function. The results indicate that nhr-6 may perform roles in both hermaphrodite gonad development and embryogenesis. In F1 progeny from injected parents, we observed high-penetrance gonadal defects, abnormal oocyte development, and a low but reproducible incidence of embryonic lethality. As has been reported for several other genes, the RNAi effects were also observed in the F2 and F3 generations. The nhr-6 temporal expression profile obtained by Northern analysis and RT-PCR is consistent with the view that nhr-6 functions in embryogenesis and gonad development. nhr-6 mRNA is present in all developmental stages, with the highest expression levels in the embryo and the adult. We have observed the first specific mutant phenotype for the conserved class of NGFI-B nuclear receptors, as knockouts of the mouse NGFI-B gene yielded apparently normal animals. The lack of an observable phenotype in the NGFI-B knockout animals is most likely due to redundancy of function with the highly similar NOR1 and Nurr1 genes. Efforts to obtain nhr-6 mutants for further characterization are underway. Additionally, the construction of an nhr-6::GFP fusion gene and the production of anti-NHR-6 antibodies for spatial expression studies are in progress.