WANG, L. et al. (2015) International Worm Meeting "A Neural Circuit of Caenorhabditis elegans for Memory-Dependent Na+ Chemotaxis."

Kunitomo, H., WANG, L., Tomioka, M., Iino, Y.

[International Worm Meeting, 2015]

Animals show various behaviors in response to environmental conditions, which are often plastic depending on previous experiences. C. elegans, which senses chemicals with a limited number of sensory neurons, is an ideal model for analyzing the role of each neuron in innate and learned behaviors. ASE neurons (ASE-Left neuron and ASE-Right neuron) play major roles for salt chemotaxis but have lateralized functions: ASER neuron is activated by decreases in NaCl concentrations and ASEL neuron is activated by increases in NaCl concentrations. So far, our lab and others have focused on the roles of ASER and its signaling pathways in salt chemotaxis plasticity, but whether and how ASEL regulates the plastic behaviors in salt chemotaxis remains less explored. In this study, chemotaxis assays and optogenetics combined with behavioral quantification with the worm tracking system were used to investigate the ASEL's roles in worms' behaviors and dissect the neural circuit underlying Na+ chemotaxis. Our results indicated that ASEL generates memory-dependent behaviors in Na+ chemotaxis: when worms were precultured in NaCl-free conditions, they showed no Na+ concentration preference, but when worms were precultured in NaCl-containing conditions, they migrated to higher concentrations of NaCl. Behavioral responses to optogenetic activation of ASEL neuron roughly recapitulated the chemotaxis responses: I) worms showed no response when they were transferred from NaCl-free preconditions to NaCl-containing conditions; II) worms showed reduction of reversal/turning frequency upon activation of ASEL when they were preconditioned in NaCl-present conditions, or when they were preconditioned in NaCl-free conditions and tested in NaCl-free conditions. The neural circuit to regulate the behavior (under condition II) was further examined and ASEL's downstream interneurons AIY, AIA, and AIB were all found to be involved in regulating the behavioral responses. Based on our results, AIY is the most important downstream interneuron of ASEL, which is activated by ASEL and responsible for stimulating forward locomotion. AIA seems also activated by ASEL and stimulate forward locomotion, while AIB is inhibited by ASEL and stimulates the reversal/turning behavior. Therefore, under condition II, all three interneurons receive signals from ASEL to stimulate forward movement and reduce turning, driving the worms to higher concentrations.

Wang, L. et al. (2019) International Worm Meeting "Glucose-induced unfolded protein response extends lifespan of aged animals."

Thalappilly, S., Beevi, Z., Thibault, G., WANG, L., Beaudoin-Chabot, C.

[International Worm Meeting, 2019]

Aging is one of the most critical risk factors for the development of metabolic syndromes. Both metabolic diseases, type 2 diabetes and insulin resistance, have a strong association with endoplasmic reticulum (ER) stress. Upon ER stress, the unfolded protein response (UPR) is activated to limit cellular damage by adapting to stress conditions and to re-establish ER homeostasis. However, genes upregulated from the UPR tend to decrease while the incidence of developing metabolic syndromes increases with age. Although stress resistance correlates with increased longevity in a variety of model organisms, the link of the UPR, ER stress resistance and longevity remains poorly understood. Here, we report that high glucose diet (HGD) extends lifespan in aged worms in contrast to reduced lifespan in young adult animals subjected to HGD. Surprisingly, our data suggest that glucose-induced longevity in aged worms works independently of the insulin/IGF-1 signalling (IIS) pathway. Aged animals subjected to HGD lived longer in a UPR-dependent manner. To restore cellular homeostasis upon ER stress, the three highly conserved UPR transducers ATF-6, IRE-1, and PEK-1 are activated by HGD. We identified that ATF-6 and PEK-1 play a role in extending longevity but not IRE-1 upon HGD. Based on these observations, we hypothesise that HGD activates the UPR in aged worms to overcome aging-associated ER stress and to restore homeostasis.

wang, l. et al. (2019) International Worm Meeting "Role of glial enriched ion channels and transporters in C. elegans sensory behaviors."

Bianchi, L., Grant, J., Encalada, N., WANG, L.

[International Worm Meeting, 2019]

Glial cells perform many functions, including providing nutrients to neurons and maintaining the extracellular concentration of ions and other solutes. In doing so, glia regulate the function of neurons and, ultimately, animal behaviors. However, the molecular mechanisms that are the basis of the regulation of neuronal output by glia are not fully understood. Our lab is using the model organism C. elegans to investigate molecular mechanisms of glia-neuron interactions. To this end, we recently sequenced the mRNA extracted from amphid sheath glia, a pair of cephalic glial cells that ensheath the dendrites of 12 pairs of sensory neurons. Among the ~1,000 glial-enriched genes, we identified 14 ion channel genes with 2.7- to 29.6-fold mRNA enrichment in glia as compared to other cells. These genes belong to all the major ion channel classes, including voltage-gated and two pore domain K+ channels, TRP channels, voltage gated Cl- channels, and cys-loop ionotropic receptors. To determine whether these channel genes are needed for sensory behaviors, we acquired knock-out strains, generated glial specific RNAi transgenic strains, and performed chemotaxis assays. Our results show differential effects of knock-out of these genes on attraction or avoidance responses to odors, such as octanol. These results suggest that homeostasis of ions and solutes have differential impact on the function of amphid sensory neurons in C. elegans, with some ions being more critical and specific to the function of certain neurons.. Taken together, this work expands on our understanding of glia contribution to C. elegans behavior via regulation of the composition of the microenvironment surrounding neurons.

Wang, Christopher L C et al. (2009) International Worm Meeting "Characterization of TEG-1 in C. elegans germ line."

Zhao, Lina, Schedl, Tim, Wilson-Berry, Laura, Wang, Christopher L C, Hansen, Dave

[International Worm Meeting, 2009]

The C. elegans germline is a polar tissue with distal cells mitotically dividing, while more proximal cells enter meiosis and differentiate into gametes. Results from biochemical and genetic studies indicate that the GLP-1/Notch signaling pathway promotes distal germ cell proliferation by antagonizing two redundant pathways, one containing GLD-1 and the other GLD-2, which each promote meiotic entry (1-3). Two genetic screens were performed to identify novel genes involved in regulating the proliferation vs. differentiation decision. In one screen, mutations that enhanced the over-proliferation phenotype of a weak glp-1(oz112oz120 gf) allele were identified. In the other screen, mutations that were synthetic tumorous with a gld-2 null allele were isolated. As a result, teg-1 (tumours enhancer of glp-1(gf)) was identified in both screens. The open reading frame of teg-1 encodes a protein of 370 amino acid residues. TEG-1-related proteins are found from yeast to human and contain GYF domain that is thought to play a role in protein-protein interactions (4). We have raised antibodies against TEG-1 peptides, which detect a band at about 65 kDa in N2 lysate, but not in a teg-1 null mutant lysate, indicating the antibodies are specific to TEG-1. Subcellular localization studies demonstrate that TEG-1 is found in all germline and somatic nuclei of the gonad, with stronger staining being detected in distal tip cell, proximal germ cells, and oocytes. Moreover, we found that TEG-1 co-immunoprecipitates with UAF-1, the large subunit of U2 small nuclear ribonucleoparitcle (snRNP) auxiliary factor (5), suggesting TEG-1 forms a complex with a subset of UAF-1. This finding is reminiscent the human TEG-1 homolog CD2BP2, which interacts with a tri-snRNP bridging protein that is a component of the spliceosome complex (6), and suggests C. elegans TEG-1 may also involve in pre-mRNA splicing. We are currently conducting experiments to identify potentially misspliced mRNAs in teg-1 mutants to better understand the mechanism of TEG-1in regulating the proliferation vs. meiotic entry decision in the germ line. 1. Hubbard, E.J. 2007. Dev. Dyn. 236: 3343-3357. 2. Kimble, J. and S.L. Crittenden. 2007. Annu. Rev. Cell Dev. Biol. 23:405-33. 3. Hansen, D. and T. Schedl. 2006. Curr. Top. Dev. Biol. 76:185-215. 4. Kofler, M.M. and C. Freund. 2006. FEBS J. 273:245-256. 5. Zorio, D.A. and T. Blumenthal. 1999. Nature 402:835-838. 6. Laggerbauer. B. et. al. 2005. RNA 11:598-608.

Kyung Won Kim et al. (2007) International Worm Meeting "RNP-8, a newly identified co-activator of GLD-2 cytoplasmic poly(A) polymerase, and its control of germline development."

Kyung Won Kim, Liaoteng Wang, Judith Kimble

[International Worm Meeting, 2007]

Polyadenylation is critical for mRNA stability and translation. In general, poly(A) tail elongation can stabilize and translationally activate mRNAs, while poly(A) tail removal can trigger mRNA degradation and repress translation. One key regulator of polyadenylation is GLD-2, a widely-conserved cytoplasmic poly(A) polymerase (PAP) (1, 2). GLD-2 proteins are catalytic subunits but have no recognizable RNA-binding domain (1). Our work focuses on GLD-2 in the nematode C. elegans and its control of germline development. Our current model is that a nematode GLD-2 is recruited to specific mRNAs by specific RNA-binding partners. GLD-2 has low PAP activity on its own, but its activity is stimulated by interactions with GLD-3/Bicaudal-C, a conserved RNA-binding protein (1). To identify other GLD-2 partners, we conducted a yeast two-hybrid screen and identified RNP-8, a novel RRM protein that is predicted to bind RNA. Consistent with our model, we find that RNP-8 stimulates GLD-2 PAP activity in vitro. Both rnp-8 mRNA and RNP-8 protein are germline-enriched and abundant in the cytoplasm of germ cells in the pachytene stage of meiotic prophase and in maturing oocytes. An rnp-8 deletion mutant exhibits low percentage of the Mog (for masculinization of germline) phenotype, suggesting that RNP-8 promotes oogenesis. Importantly, its minor phenotypic defects are enhanced by removal of other RNA regulators controlling germline development. 1. Wang, L., Eckmann, C.R., Kadyk, L.C., Wickens, M. and Kimble, J. (2002) A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316. 2. Kwak, J.E., Wang, L., Ballantyne, S., Kimble, J., and Wickens, M. (2004) Mammalian GLD-2 homologs are poly(A) polymerases. Proc Natl Acad Sci USA 101, 4407-4412.

Kyung Won Kim et al. (2005) International Worm Meeting "RNP-8 interacts with the GLD-2 cytoplasmic poly(A) polymerase and regulates its polyadenylation activity"

Kyung Won Kim, Judith Kimble, Liaoteng Wang

[International Worm Meeting, 2005]

Polyadenylation is critical for mRNA stability and translational activation. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs promotes their translation. The C. elegans gld-2 gene is required for multiple steps in germline development, including the mitosis/meiosis decision. GLD-2 is a cytoplasmic poly(A) polymerase (PAP) that lacks an RNA recognition motif (1); similar PAPs have been identified in virtually all eukaryotic organisms (2). A yeast two-hybrid screen using GLD-2 as bait isolated GLD-3, RNP-8 and LARP-1 (1; L. Wang and J. Kimble, unpublished). GLD-2 has little PAP activity on its own, but it is stimulated in vitro by GLD-3 (1). We have now focused on RNP-8 to ask whether this GLD-2 interactor also stimulates GLD-2 activity and to determine whether RNP-8 has a major role in germline development. Preliminary data reveal that rnp-8(RNAi) and gld-2 mutants have similar defects during oogenesis. Both gld-2 and rnp-8 mRNAs are abundant in embryos, L4 larvae, and adults, and rnp-8 mRNA is enriched in the germ line. To test whether RNP-8 stimulates the GLD-2 PAP activity, we performed assays for PAP activity in vitro. Preliminary data suggest that RNP-8 can indeed stimulate GLD-2 PAP activity. Taken together, we suggest that RNP-8 stimulates GLD-2 activity to control germline development.1. Wang, L., Eckmann, C.R., Kadyk, L.C., Wickens, M., and Kimble J. (2002), A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316 2. Kwak, J.E., Wang, L., Ballantyne, S., Kimble J. and Wickens, M. (2004), Mammalian GLD-2 homologs are poly(A) polymerases. PNAS 101: 4407-4412

Hunter CP et al. (1991) International C. elegans Meeting "NON-AUTONOMY OF HER-1 FUNCTION IMPLICATES CELL INTERACTIONS IN C. ELEGANS SEX DETERMINATION."

Hunter CP, Wood WB

[International C. elegans Meeting, 1991]

Activity of the her-l gene is required for normal male development in C. elegans. Loss-of-function her-l mutations cause feminization of XO animals (normally male) to produce fertile hermaphrodites, while gain-of-function her-l mutations cause masculinization of XX animals ( normally hermaphrodite) to produce pseudo-males. Genetic analysis indicated that her-l functions early in the hierarchy of regulatory interactions among the major sex determination genes (1). We have analyzed her-l genetic mosaics to determine which cells must express her-l for wild-type male development. If her-l functions cell- autonomously, then in XO animals her-l (+) cells will follow male fates, and her-1(-) cells will follow hermaphrodite fates. If her-l or a her-l regulated activity functions non-autonomously, then the her-l genotype of a cell may not determine its sexual phenotype. The phenotypes of XO her-l genetic mosaics are variable, but clearly show that both her-1(-) and her-l (+) cells can follow either hermaphrodite or male fates. We conclude that her-l is neither necessary nor sufficient to cell-autonomously determine male sexual phenotypes and, therefore, that her-l or a her-l regulated activity must be able to function non-autonomously. The observed patterns of non-autonomous effects suggest that many or all cells express and respond to her-l activity. These findings implicate cell interactions in C. elegans sex determination. Earlier genetic analysis indicated that her-l might directly control tra-2 activity (1). Molecular characterization of the gene products of her-l and tra-2 (see abstracts by M. Perry et al. and P. Kuwabara et al) is consistent with a model in which a secreted ligand encoded by her-l interacts with a cell-surface receptor encoded by tra-2. We will discuss the possible role of such an interaction in coordinating the sex determination decision.

Ellis RE et al. (1991) International C. elegans Meeting "PROGRESS IN CLONING fog- 1"

Kimble JE, Ellis RE

[International C. elegans Meeting, 1991]

The fog-l gene plays an important role in the decision of germ cells to differentiate as either spermatocytes or oocytes (Doniach 1986, Barton and Kimble 1990). In fog-l mutants, all germ cells develop into oocytes. This is true for both males and hermaphrodites. However, these fog-l mutations do not appear to affect other aspects of sexual development. Furthermore, the fog-l mutant phenotype is not suppressed by mutations in any of the other genes known to control sex- determination. Thus fog-l might function to initiate spermatogenesis in response to other genes that regulate general sexdetermination. There are 42 fog-l mutations. These mutations arise at a frequency typical for knockout mutations in other C. elegans genes, and four of these fog-l alleles were isolated from a screen capable of recovering deletions. Thus it is likely that these mutations cause a loss of fog- l function. In order to study the mechanisms by which fog-l functions, and to determine how fog-l expression is regulated by the genes that control sex-determination, we are now cloning this gene. The fog-l gene is located on the left arm of LGI, between sup-ll and unc-ll, a region spanned by a large contig of about one megabase in size. By mapping DNA polymorphisms with respect to fog-l, we have narrowed down the region in which fog-l must be located, and are now injecting YAC DNA that covers this region in an attempt to rescue fog-l mutants.

Lackner MR et al. (1995) International C. elegans Meeting "The role of mpk-l in vulval development"

Kim SK, Lackner MR

[International C. elegans Meeting, 1995]

The mpk-l gene encodes a MAP kinase protein that is approximately 75% identical to vertebrate MAP kinases. Previously, we have shown that mpk-l plays an important role in ras-mediated induction of vulval cell fates, as reduction-offunction mutations in mpk-l completely suppress excess vulval differentiation caused by a gain-of-function mutation in let-60 ras. We have now characterized a strong reduction-offunction allele of mpk-l that results in temperature-sensitive vulvaless phenotype (84% Vul at 25 degrees Celsius) and a completely penetrant sterile phenotype at all temperatures. Epistasis analyses suggest that mpk-l acts downstream of lin-15 and let-60 ras, but upstream of the putative transcription factors lin-l and lin-31. To determine whether mpk-l is absolutely essential for vulval induction, or whether some induction may occur in the absence of mpk-l activity, we are in the process of isolating a null allele of mpk-l. We used mutant activated forms of mpk-l and Drosophila MEK to address the question of whether activation of these two kinases is sufficient to promote vulval fates in the absence of upstream signaling. Coexpression of both activated mpk-l and activated D-MEK causes a strong Muv phenotype (~90% Muv), whereas coexpression of wild-type mpk-l and wild-type D-MEK results in a weak Muv phenotype (~10% Muv). The strong Muv phenotype is unaffected by ablation of the anchor cell or a reduction-offunction mutation in lin-45 raf, consistent with activation of mpk-l and MEK being at least partially independent of upstream signaling. Our future plans are to identify targets of mpk-l by isolating suppressors of this strong Muv phenotype.

Mango SE et al. (1991) International C. elegans Meeting "A NOVEL glp-1 ALLELE MIMICS THE EFFECT OF lin-12(d) ON VULVAL DEVELOPMENT."

Kimble JE, Maine EM, Mango SE

[International C. elegans Meeting, 1991]

The lin-12 and glp-l genes encode homologous transmembrane proteins ( l) that may act as receptors for cell interactions during development ( 2,3). The glp-l gene is required zygotically for germ line proliferation and maternally for embryogenesis (2, 4); lin-12, in contrast, is involved in various postembryonic cell interactions in the soma, including those between vulval precursor cells (VPCs) that specify vulval fates (5). The novel allele glp-l (q35) has a semidominant Multivulva phenotype (Muv) in addition to the typical recessive Glp phenotypes. We find that the Muv phenotype of glp-l (q35) is similar to that of lin-12(d) in three ways. First, VPCs that would normally fuse with the hypodermal syncytium (3 fate) follow a 2 -1ike vulval lineage instead. Second, the Muv phenotype is not dependent on the anchor cell. Third, the Muv phenotype results from increased or novel, rather than decreased, glp-l activity. Because glp-l (q35) remains Muv in animals lacking lin-12, this effect does not depend on lin-12 activity. We conclude that the glp-l (q35) product is functionally very similar to that of lin-12. The glp-l (q35) gene bears a nonsense mutation predicted to truncate the glp-l protein 122 amino acids from its carboxy terminus. The region which should be missing from the glp-l (q35) protein includes a PEST sequence thought to destabilize proteins. A simple explanation of these results is that the carboxy terminus bears a negative regulatory domain that normally keeps glp-l activity off in the VPCs. In glp-l ( q35) animals, inappropriate glp-l activity may drive these cells to proliferate, and thereby channel them toward a 2 -like fate.