Xu, Ningyi et al. (2011) International Worm Meeting "Local triglyceride synthesis by DGAT-2 promotes lipid droplet expansion."

McKinney, Sean, Cole, Ronald, Zhang, Shaobing, Xu, Ningyi, Guo, Fengli, Mak, HoYi

[International Worm Meeting, 2011]

Lipid droplets (LDs) are evolutionarily conserved organelles for cellular fat storage. We have previously shown that a loss of DAF-22 function, which attenuates peroxisomal fatty acid beta-oxidation, causes LD expansion and an increase in triglyceride levels. In order to identify genes that are required for LD expansion, we performed a genetic suppressor screen in the daf-22 mutant background and recovered multiple alleles that fell into at least 5 complementation groups. We report here the molecular cloning of dgat-2, which encodes a conserved diacylglycerol acyltransferase, the terminal enzyme for triglyceride synthesis. Loss of dgat-2 function inhibits LD expansion and reduced triglyceride levels of daf-22 mutant animals while over-expression of DGAT-2 is sufficient to promote LD expansion in wild-type animals. Photo-bleaching demonstrates that DGAT-2 is a LD resident protein. Electron microscopy reveals that DGAT-2 clusters at discreet foci on the LD surface. We engineered a mutant DGAT-2 that was tethered to the ER. This mutant DGAT-2 failed to support LD expansion, indicating that LD localization of DGAT-2 is necessary for its proper function. Our results demonstrate that local synthesis and deposition of triglyceride is critical for LD expansion. We are currently searching for proteins that act in concert with DGAT-2 using genetic and proteomic approaches.

Singh, Varsha et al. (2021) International Worm Meeting "Neuronal control of lipid metabolism by STR-2 G protein-coupled receptor promotes longevity in C. elegans"

Dixit, Anubhuti, Shashikanth, Meghana, Koushika, Sandhya, Singh, Varsha, Modi, Souvik, Sandhu, Anjali, Watts, Jennifer

[International Worm Meeting, 2021]

The G proteincoupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. We find that C. elegans longevity is regulated by a chemosensory GPCR STR2, expressed in AWC and ASI amphid sensory neurons. STR2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR2 regulates expression of delta9 desaturases, fat5, fat6 and fat7, and of diacylglycerol acyltransferase dgat2. Rescue of the STR2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat5, dgat2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wildtype levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.

Tran, Tra My et al. (2021) International Worm Meeting "Influences on fat content, fat density and lipid droplets in C. elegans liquid culture"

Schonfelder, Gilbert, Oelgeschlager, Michael, Vogl, Silvia, Wagner, Aline, Menzel, Ralph, Tran, Tra My, Steinberg, Gianna

[International Worm Meeting, 2021]

C. elegans has become an emerging model system to investigate different toxicological endpoints. In particular, fundamental pathways regulating energy homeostasis are highly conserved between C. elegans and humans and thus, the worm provides an important model for the analysis of mechanisms leading to obesity. For example, the analysis of lipid homeostasis as well as fat accumulation in C. elegans can provide important insights into metabolic diseases and the activity of potential obesogenes. In addition, changes in lipid homeostasis by external factors are suspected to be transmitted to subsequent generations. Thus, lipid metabolism might also be a promising read-out in C. elegans to address inter- or transgenerational effects of xenobiotics. In a first step, we studied the influence of different food regimes on worm development, fat content and changes on the level of lipid droplets (LD), the worm's major form of fat storage, to generate "low body fat" or "high body fat" phenotypes. We exposed C. elegans to different OP50 concentrations in liquid culture for three days (L1 to adulthood) and measured worm sizes, triacylglyceride (TAG) contents, LD sizes and density as well as fatty acid (FA) patterns. After three days, worms fed with higher OP50 concentrations showed an increase in body size, higher TAG levels and significant increases in LD size and volume per microm3 in the anterior part of the intestine. Moreover, GC-MS analysis revealed significant differences in FA pattern of adult worms and their eggs, when comparing the "fat" and "lean" phenotypes. Notably, cyclic FA and polyunsaturated fatty acids (PUFA) were affected in a diet-dependent manner. Interestingly, orlistat, an anti-obesogenic drug, could also influence fat density and FA patterns in adult worms as well as eggs. Currently, we are testing effects of (anti-)obesogenes on fat content and FA composition and if those are transmitted to subsequent generations to assess the applicability of these read-outs for the analysis of inter- or transgenerational (adverse) effects.

WB Derry et al. (1999) International C. elegans Meeting "Analysis of the C. elegans homolog of the FHIT tumor suppressor gene"

WB Derry, S van Kreeveld, JH Rothman

[International C. elegans Meeting, 1999]

Alterations in the FHIT gene occur frequently in the development of several human cancers (1). The Fhit protein is a diadenosine P 1 , P 3 -triphosphate hydrolase and is a member of the histidine triad superfamily of nucleotide binding proteins (2). The cellular mechanism of Fhit activity and the relationship between Fhit signaling and tumorigenesis are presently unknown. The C. elegans and Drosophila FHIT genes encode a fusion protein in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases, and are referred to as NitFhit (3). We are interested in understanding the role of NitFhit in development and programmed cell death. RNAi of C. elegans NitFhit causes an embryonic arrest phenotype, suggesting an essential role for this gene in development. We are currently analyzing the loss-of-function phenotype and the effect of ectopic NitFhit expression on viability and programmed cell death in the worm. (1) Huebner, K., Garrison, P.N., Barnes, L.D. & Croce, C.M. (1998). Ann. Rev. Genet ., 32 : 7-31. (2) Barnes, L.D., Garrison, P.N., Siprashvili, Z., Guranowski, A, Robinson, A.K., Ingram, S.W., Croce, C.M., Ohta, M. & Huebner, K. (1996). Biochemistry , 35 : 11529-11535. (3) Pekarsky, Y., Campiglio, M., Siprashvili, Z, Druck, T., Sedkov, Y, Tillib, S., Draganescu, A., Wermuth, P., Rothman, J.H., Huebner, K., Buchberg, A.M., Mazo, A., Brenner, C. & Croce, C.M. (1998). Proc. Natl. Acad. Sci. USA , 95 : 8744-8749.

Tetsu Saigusa et al. (2003) International Worm Meeting "C. elegans as a novel model system for the molecular study of circadian clock."

Tetsu Saigusa, Masatoshi Hagiwara, Kenji Hasegawa, Naoaki Ishii, Yoshishige Kimura, Akihiro Tanakadate

[International Worm Meeting, 2003]

Transcriptional/translational feedback loops between clock genes such as per and tim (first cloned in Drosophila) and their protein products are thought to constitute the core of the circadian clock. C. elegans has been known to inherit a per homologue, lin-42. Levels of lin-42 mRNA oscillate synchronously with approximately 6-hr molting cycle at the larvae stage and become flat at the adult stage. We have succeeded to show a convincing circadian rhythm of the locomotor activity in adult C. elegans, using the automatic device (Bug-tracker) to record footprints of an isolated animal. The rhythm exhibit characteristics such that: 1) the nematode in the larva stage exhibit a behavioral rhythm with a period shorter than 24 hours ; 2) the rhythm in the adulthood can be entrained at a 12-h light and 12-h dark cycle (LD) ; 3) the rhythm free runs with the period of about 24 hour in the range of temperature between 15 and 25 degrees C in constant dark (DD) ; 4) the rhythm is entrained to 180 degrees reversed LD(=DL) with repeated L phase or D phase on an LD. These result prove that the nematode inherits a convincing circadian clock with caracteristics common to the circadian clocks of other animal. Also suggest a possibility that per and relating clock genes are not indispensable for the circadian rhythm in the animal kingdom and that further universal system should lie behind, which should even governs the clock genes. To identify the universal circadian system, we are examining the behavioral rhythms in nematodes with knockout and/or rescued genes considerable to be involved in the circadian timekeeping, using a newly devised system with multi-channel, which enables us to measure the circadian behavioral rhythms of a number of isolated nematodes at once. The nematode will provide a novel excellent model for the molecular study of circadian clock.

Asher D Cutter (2003) International Worm Meeting "How many sperm are enough?"

Asher D Cutter

[International Worm Meeting, 2003]

Sperm limited fecundity in selfing C. elegans hermaphrodites has been explained to be the consequence of a trade-off between selection for greater total fecundity and more rapid turnover of generations. Greater numbers of sperm increase the total number of progeny produced by a hermaphrodite, but delay the onset of fertilization and therefore reduce the intrinsic rate of population growth. Both empirical and theoretical work support this general explanation (Hodgkin & Barnes 1991; Barker 1992). However, the question remains: can this simple model explain the actual numbers of sperm produced by C. elegans hermaphrodites? In this study, I extend a well-known population model to calculate the expected number of sperm that hermaphrodites would produce to maximize fitness (i.e. the intrinsic rate of growth). I apply literature-based and new empirical estimates of rates of gamete production and the fraction of sperm produced during larval development to the model. I find that the model predicts sperm counts consistent with observed sperm numbers in C. elegans hermaphrodites, given available empirical data, and provided that precocious larval production of sperm is taken into account. Several testable hypotheses follow from the model regarding how natural selection and environmental variation may influence sperm production among populations or among species with a similar mode of reproduction. J. Hodgkin & T.M. Barnes. 1991. Proc. R. Soc. London B. 246: 19-24; D.M. Barker. 1992. Evolution. 46: 1951-1955.

Li-Wei Lee et al. (2007) International Worm Meeting "Expression of hepatitis D viral antigen by bicistronic construct in Caenorhabditis elegan."

Chi-Ruei Huang, Li-Wei Lee, Hsiao-Wen Lo, Keng-Poo Tan, Szecheng J. Lo

[International Worm Meeting, 2007]

To investigate whether a viral protein which is dominate present in the nucleolus will affect the nucleolus structure of Caenorhabditis elegans, we developed a bicistronic construct derived from C. elegans operon CEOP5428 containing (fib-1) fibrillarin and rps-16 genes. When a construct containing pfib-1::GFP-rps-16::Cheery was expressed in transgenic worms, both GFP and cheery signal were detected. We then substituted the rps-16::cheery into DesRed::LD (large antigen of hepatitis D viral antigen) and obtained transgenic worms expressing both signals in the nucleus. The preliminary results showed that no significant phenotypes appeared in transgenic worm. However, this bicistronic construct can be employed to express different uncharacterized viral genes or oncogenes for studying their effect on phenotype as a quick functional screen.

Joseph A. Ross et al. (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "High-density genotyping in Caenorhabditis briggsae advanced-intercross recombinant inbred lines reveals unexpected inter-strain genomic incompatibilities"

Julia E. Staisch, Daniel C. Koboldt, Joseph A. Ross, Bhagwati P. Gupta, Scott E. Baird, Raymond D. Miller, Helen M. Chamberlin, Eric S. Haag

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

Caenorhabditis briggsae is a useful species with which to pursue evolutionary and genomic questions by comparison with C. elegans. However, in C. briggsae the genetic maps and genome assembly necessary for robustly conducting experiments, such as mapping mutations, are not of comparable quality to those available for C. elegans. To improve the genetic map and genome assembly of C. briggsae, 180 AF16xHK104 and HK104xAF16 advanced-intercross recombinant inbred lines (AI-RIL) were genotyped at 1,536 single nucleotide polymorphism (SNP) markers using the Illumina GoldenGate platform. Following quality control, the final dataset comprised 167 AI-RIL typed at 1,034 markers. Employing AI-RIL increased the number and decreased the size of haplotype blocks per chromosome, and the dense panel of genetic markers allowed most previously unplaced sequence contigs to be ordered within chromosome assemblies. Additional issues with the present genome assembly, including contig misassignment to chromosomes, have been resolved to produce a new genome assembly (cb4). Our genotyping data also reveal interesting population genetic phenomena. Analysis of parental allele fraction of the AI-RIL reveals strong bias of three autosomes in favor of the HK104 parental allele. The bias of one autosome is independent of cross direction and due to a slow-growth phenotype elicited in a hybrid genome. The bias of the other two autosomes is strongly dependent on cross direction. Comparison of inter-chromosomal linkage disequilibrium (LD) in the AI-RIL also reveals the presence of cross-direction-specific LD. Together, the genome-wide study of marker segregation in C. briggsae AI-RIL reveals evidence of inter-strain genomic incompatibilities that suggest the onset of genomic divergence and potentially incipient speciation. Efforts are currently under way to identify the incompatibility loci involved.

Takagi S et al. (1995) International C. elegans Meeting "THE GENE MAU-2 IS REQUIRED FOR CELL AND AXONAL MIGRATION ALONG BOTH BODY AXES"

Hekimi S, Shioi G, Takagi S

[International C. elegans Meeting, 1995]

mau genes are defined by maternal effect unc genes. In mau mutants, the progenies from homozygous mothers are Unc , but those from heterozygous mothers are not. This mode of inheritance suggests that some mau genes are involved in the development of the nervous system. mau-2 animals show a Kinky-Unc and Egl phenotype. We have analyzed the neuroanatomy of mau-2 mutant worms by making use of 1) animals arrested at larval stages ("living-dead") in which neurons and other structures of the body wall are clearly visible, and 2) the expression of reporter genes (lacZ,GFP) under the control of neuron specific promoters. We have found that the neurons that are known to undergo long-range migration during the embryonic and larval development are sometimes mispositioned in respect to both body axes. In addition, the dorsal cord and other longitudinal axon bundles are often defasciculated. Thus mau-2 gene appears to be required for cell and axonal migrations during development (see also abstract by Benard, Takagi, Barnes and Hekimi).

Kong X et al. (1996) East Coast Worm Meeting "Characterization of a gene whose expression is dependent on unc-24"

Ma C, Kong X, Chalfie M

[East Coast Worm Meeting, 1996]

Worms with mutations in the unc-24 gene have difficulties moving forward. An opposite phenotype is caused by mutations in the unc-4 gene. The UfZC4 animals can move forward but can not move backward. Using a novel PCR method to construct difference libraries, we have previously identified 18 cDNAs that has reduced expression in the unc-24(e448) background. The unc-24 gene has been cloned by Tom Barnes and it encodes a stomatin-like protein (Barnes et al. J. Neurochemistry, in press). Our results suggest that unc-24 may be involved in a signal transduction pathway that regulates gene expression. We are particularly interested in a clone that has decreased expression in the unc-24(e448) background but increased expression in the unc- 4(el20) background. We had tried to use the original cDNA fragment obtained from the difference library (approximately I 80bp) as the probe to screen Peter Okkema's mixed stage cDNA expression library. However, no positive clone was identified after greening more than 10 million plaques. Therefore, we believe that the cDNA clone is probably not present in the cDNA library. We then adopted the procedure for both 5' and 3' RACE using the limited amount of sequence information we have from the original clone. We obtained multiple cDNA clones that encompass the full coding sequence of this gene. The longest cDNA is 424bp in length and it encodes a small 91 aa protein. Interesting, an alternatively spliced form is also found that spliced out 50 bp of coding sequence, producing a truncated protein that has a shorter C-terminus. The protein contains a RGD sequence and the C-terminus is predicted to have a GPI-anchored sequence. The smaller protein does not contain the GPI anchored signal so it might be secreted instead of anchored on the membrane. The protein is then likely to be involved in some cellcel1 adhesion process. The sequence has no match in the existing C. elegans genome database and we will determine the physical location of this gene using Yac grid. We have also obtained a genomic clone of this gene that contains approximately 2.5 kb of upstream sequence from the Lamda FixII genomic library. We are making reporter fusions with this gene to determine its expression pattern and to screen Hengartner lab's transposoninsertion bank for Tc1 insertion mutations. In addition, we will try to determine whether the expression of this gene is affected in other unc-24 alleles as well as other known forward unc mutants such as unc-l, unc-7, unc-9, and unc-23.