Mello, D.F. et al. (2019) International Worm Meeting "Linking mitochondrial dysfunction during development with pathogen resistance later in life."

Baugh, R.L., Mello, D.F., Meyer, J.N., Bergemann, C.M.

[International Worm Meeting, 2019]

Growing evidence has shown that mitochondrial dysfunction not only compromises the energetic metabolism of cells, but also plays key roles in other physiological processes such as immunomodulation. We hypothesize that mitochondrial toxicity can be a common link between increased prevalence in immune-related disorders and toxic environmental exposures. To test this hypothesis, we are using the pesticide rotenone -a widely known complex I inhibitor- and the model organism Caenorhabditis elegans. Synchronized N2 eggs were exposed to rotenone (0-0.5 M) or 0.25% DMSO (control) in liquid with food (HB101) and harvested 52h later (L4 stage). After a further 48h depuration period, worm survival was followed in the presence of the pathogens Pseudomonas aeruginosa strain PA14, and Salmonella enterica serovar Typhimurium strain SL1344. Our first finding was that rotenone caused a dose-dependent decrease in worm size, which was associated to developmental delay. Worm vulval development was assessed to precisely determine the hours of developmental delay. Stage-synchronized worms exposed to 0.5 M rotenone had a longer median survival in SL1344 than control animals (~40%); but were more susceptible to PA14 (~15%). To validate whether these altered pathogen responses were due to rotenone-induced mitochondrial dysfunction, we analyzed different mitochondrial parameters. No significant differences were observed in preliminary measurements of worm basal oxygen consumption rate (OCR), spare capacity and ATP-linked OCR, or the ratio of mitochondrial to nuclear DNA copy number. This apparent lack of mitochondrial dysfunction after a developmental rotenone exposure may be due to a metabolic compensation in the worms, most likely through upregulation of complex II activity and the glyoxylate cycle, according to previous work. Thus, this appears to be a great model to study signaling between mitochondria and the immune system caused by metabolism shifts, without the detrimental effects of overt mitochondrial dysfunction. Now we are investigating the mechanisms by which mitochondrial signaling might be regulating the observed shifts in resistance to pathogens.

Sinniah B (1979) Southeast Asian J Trop Med Public Health "Parasites of some rodents in Malaysia."

Sinniah B

[Southeast Asian J Trop Med Public Health, 1979]

A total of 2,337 rodents trapped from various parts of Peninsular Malaysia were dissected and studied for the distribution and prevalence of parasitic infections. Four new rodent hosts for Sarcocystis in Malaysia are reported (Bandicota indica, Rattus sabanus Rattus argentiventer and Rattus norvegicus). Sarcocystis was found in 17.2 percent of the rodents examined. Rattus annandalei, Rattus tiomanicus and Rattus norvegicus are new hosts of Syphacia muris in Peninsular Malsysia. Rattus sabanus was found to be infected with Zonorchis borneonenis. Brachylaima ratti Baugh, 1962 was recovered from the small intestine of Rattus rattus diardii for the first time in Malaysia. The prevalence and distribution of other parasites are also discussed.

Joanne C Wen et al. (2003) International Worm Meeting "Functional Characterization of Early Embryonic PAL-1 Targets"

Craig P Hunter, Joanne C Wen, L Ryan Baugh

[International Worm Meeting, 2003]

The activation and maintenance of C lineage specification occurs through maternal and zygotic PAL-1 activity, respectively (Hunter & Kenyon, 1996; Edgar et al, 2001). A set of targets of this master regulatory transcription factor were identified by transcript profiling embryos with perturbed PAL-1 activity (see abstract by Baugh et al). To functionally characterize PAL-1 targets, we have used RNAi to assess the lethality and terminal phenotypes following loss of function. To identify interactions between targets, we are performing epistasis analysis both by scoring synthetic lethality and by examining the effect of RNAi against one target on the expression of reporters for other targets. Our hope is that such functional characterization of a key set of PAL-1 targets will generate the data necessary to begin modeling the PAL-1 regulatory network.

Julia M Claggett et al. (2004) East Coast Worm Meeting "An Investigation into the potential regulatory relationships between components of a network specified by pal-1."

L Ryan Baugh, Julia M Claggett, Craig P Hunter

[East Coast Worm Meeting, 2004]

PAL-1 protein, contributed both maternally and zygotically, is necessary and sufficient to specify and maintain the C blastomere lineage in the C. elegans embryo (Hunter and Kenyon, 1996). A number of this master regulator's targets were identified by microarrays comparing the transcript abundance in wild-type and mutant embryos either lacking or containing extra C blastomeres. Furthermore, we collected these embryos at defined time points, thus additionally providing temporal information. Target genes could then be separated by their transcriptional initiation into four consecutive temporal phases defined by a singular cell cycle beginning with the 2C-cell stage (Baugh et al, 2003). Using reporter YFP constructs for thirteen of the targets and a volume-rendering program, the 3D spatial expression pattern of each target gene was established. On the basis of this spatial information and knowledge of the temporal phase to which each target belongs, we have proposed a set of regulatory relationships between the components. We are currently testing these hypotheses by disrupting potential (capital O, grave accent)upstream(capital O, acute accent) regulators via RNAi and/or mutation and either observing the effect on individual (capital O, grave accent)downstream(capital O, acute accent) reporters or analyzing the effect on transcript abundance using QPCR. We hope that such measurements will give us insight into how the genes within the pal-1 network regulate each other in order to establish and maintain the various cell fates within the C blastomere lineage. Hunter, C.P. and Kenyon, C. (1996). Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos. Cell 87, 217-26. Baugh, L.R., Hill, A.A., Slonim, D.K., Brown, E.L. and Hunter, C.P. (2003). Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome. Development 130, 889-900.

Schwarz EM et al. (1996) East Coast Worm Meeting "Cellular and molecular analysis of thermosensation"

Chalfie M, Schwarz EM

[East Coast Worm Meeting, 1996]

While senses like sight, hearing, and mechanosensation are becoming well understood, thermosensation remains obscure. Work by Hedgecock (1) and Mori (2) has shown that C. elegans is a promising model system for metazoan thermosensation, but neither the cells nor the genes required for thermosensation in C. elegans are fully known. Previous work in this laboratory (3) has shown that at least three new deg mutations cause cells in the head to degenerate while partially crippling thermosensation; none of these are allelic to previously described ttx mutations . We have therefore begun to determine which cells are defective in these deg mutations. We have also begun genetic mapping experiments aimed at positional cloning of the wild-type deg loci. References: 1. Hedgecock, E.M. and Russell, R.L. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 4061-4065. 2. Mori, I. and Ohshima, Y. (1995). Nature 376, 344-348. 3. Treinin, M. and Chalfie, M. (1993). International C. elegans meeting abstracts, p. 446.

Dasgupta, Madhumanti et al. (2020) MicroPubl Biol

Singh, Varsha, Shashikanth, Meghana, Dasgupta, Madhumanti, Bojanala, Nagagireesh

[MicroPubl Biol, 2020]

Caenorhabditis elegans feeds on bacteria in decomposing vegetation. Lipids, carbohydrates and proteins derived from microbes are digested into fatty acids, simple sugars and amino acids in C. elegans alimentary canal and absorbed by intestinal cells containing microvilli. Approximately 80% of fatty acids in C. elegans is derived from E. coli (Perez and Van Gilst, 2008). Nutrient limiting conditions can cause developmental delay in larvae (Cassada and Russell, 1975; Golden and Riddle, 1982) while complete starvation leads to L1 larval arrest or dauer formation (Baugh, 2013). Interestingly it has been reported that C. elegans fed on yeast Cryptococcus curvatus show developmental lag (Sanghvi et al., 2016) and growth arrest on Gram-positive bacterium Enterococcus faecalis (Garsin et al., 2001). We have recently shown that E. faecalis infection causes lipid droplet utilization in adult C. elegans, a process termed immunometabolism (Dasgupta et al., 2020). In this study, we have investigated the developmental arrest induced by E. faecalis in C. elegans larvae to show that the arrest is induced at L1 and L2 larva stage.

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

Gal Haspel et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Stretch Receptors and Pattern Generation in C. elegans Locomotion Circuit"

Anne Hart, Gal Haspel

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The anatomy of the nervous system of the nematode C. elegans has been comprehensively described compared to other animal species; the location and connectivity of each neuron are known. This affords us the opportunity to address the neural circuitry underlying various behaviors such as locomotion. Only five pairs of interneurons and six groups of motoneurons have been are central to locomotion based on ablation studies. Yet how, these neurons orchestrate their activity during locomotion is unclear. The sinusoid motion locomotive pattern might rise from a sensory feedback loop or involve a central pattern generator. Even in the latter case, sensory feedback might serve to adjust the pattern generator to environmental cues. Taking advantage of C. elegans optical transparency and the relative ease of genetic manipulation, we have used cameleon (a genetically encoded calcium indicator) to record the activity in specific motoneurons during fictive locomotion. Two initial findings stand out. First, mechanically moving the nematode activates motoneuron activity- suggesting the presence of stretch receptors. This supports a hypothesis first suggested by R.L. Russell based on motoneuron anatomy 30 years ago. Second, by presenting an aversive stimulus, we were able to trigger rhythmic activity in the motoneurons of immobilized animals, indicating the presence of a central pattern generator, active even in the absence of sensory feedback. These findings begin to unravel the characteristics of the nematode locomotion circuit. Understanding the orchestrated activity of neurons within this circuit will provide us with general insights into how neurons produce rhythmic movements in animals and into nematode behaviors that involve locomotion.

Stegeman, Gregory et al. (2011) International Worm Meeting "Variation in temperature-dependent behaviours among natural isolates of Caenorhabditis briggsae."

Shin, Jiwon, Ryu, William, Cutter, Asher, Bueno De Mesquita, Matthew, Stegeman, Gregory, Lin, Nan

[International Worm Meeting, 2011]

Natural genetic variation allows the discovery of new gene functions and novel alleles for genes already known to act in biologically important processes. We are applying this approach to temperature-dependent behaviours in nematode worms in order to better understand the genetics behind behaviour. We focus on Caenorhabditis briggsae because most wild caught individuals fall into two genetically distinct clades that correspond approximately with northern temperate or with tropical latitiudes. Interestingly, strains from the tropical clade have higher fecundity when reared at higher temperature than do the temperate strains, suggesting local adaptation to climate variables like temperature (Prasad et al. 2011). Movement through its thermal landscape is the main way for nematodes like C. briggsae to regulate body temperature, so we also expect to see heritable differences in temperature-dependent behaviours. Here we quantify for the first time classic thermal-response behaviours among several C. briggsae wild strains from different haplotype groups using assays like accumulation on a linear thermal gradient, isothermal tracking, and a new droplet based thermal gradient assay. We demonstrate that C. briggsae shows thermotaxis and isothermal tracking similar to C. elegans but with some differences. We also identify heritable differences among strains from wild genetic backgrounds within C. briggsae. We will continue to develop higher throughput assays for temperature-dependent behaviour in order to carry out a quantitative trait loci mapping project using recombinant inbred lines derived from tropical and temperate parental strains. Prasad, A., M. Croydon-Sugarman, R.L. Murray & A.D. Cutter. 2011. Temperature-dependent fecundity associates with latitude in Caenorhabditis briggsae. Evolution. 65: 52-63.

Romain Levayer et al. (2006) Development & Evolution Meeting "Identification of new substrates for the DYRK kinase MBK-2"

Geraldine Seydoux, Michael Stitzel, Romain Levayer

[Development & Evolution Meeting, 2006]

In C. elegans, the transition from oocyte-to-embryo requires degradation of oocyte proteins, including the oocyte maturation protein OMA-1 and the katanin subunit MEI-1. The DYRK kinase MBK-2 has been identified as a key regulator of these degradation events (Pellettieri et al., 2003). MBK-2 phosphorylates OMA-1 and MEI-1 on PEST sequences to mark these proteins for degradation (Nishi and Lin 2005, Stitzel et al. 2006, Shirayama et al., 2006). Perdurance of OMA-1 and MEI-1 explains some, but not all, of the phenotypes of mbk-2 mutants, suggesting that MBK-2 has additional substrates (Pellettieri et al., 2003). Here we describe a bioinformatic approach to identify additional MBK-2 substrates, based on properties common to OMA-1 and MEI-1. We screened a list of 4168 candidate genes, identified as "Strictly Maternal" or "Maternal Degradation" in an early embryo transcriptome analysis (Baugh et al. 2003), for: DYRK kinase consensus site PEST degradation motifs overlapping the DYRK site Embryonic lethal/sterile phenotype as reported in Wormbase Evolutionary conservation of the DYRK site Following these criteria, we identified 69 potential MBK-2 substrates. We are currently testing selected candidates for 1) phosphorylation by recombinant MBK-2 in vitro, and 2) MBK-2-dependent degradation using GFP reporters in vivo. We will report our results at the meeting.