Mahloch, A.B. et al. (2019) International Worm Meeting "Characterization of Two Genes Expressed in Chemosensory Amphid Sheath Cells."

Carstensen, H.R., Hong, R.L., Mahloch, A.B.

[International Worm Meeting, 2019]

The association between Pristionchus pacificus and its beetle host is likely mediated by the beetle pheromone, ZTDO. The mutant Ppa-obi-1(tu404) exhibited a loss of chemoattraction to ZTDO as adults and hypersensitivity to ZDTO as J4 larvae. Ppa-obi-1 is expressed predominantly at the J4 larval stage in the chemosensory glial cells known as amphid sheath cells. Amphid sheath cells are known as neuronal support cells and are essential for neuronal development and function in C. elegans. However, it is not clear if glial cells are important for the mediation of ZTDO in P. pacificus. To better understand the role of amphid sheath cells in processing ZTDO, we performed amphid sheath-specific rescue of obi-1 and found a modest recovery of J4 hypersensitivity to ZTDO. We also sought to determine if other mutations can modify the Ppa-obi-1 phenotype by using CRISPR/Cas9 to target mutations against two other genes highly expressed in glial cells: Ppa-C06E8.5(PPA25486) and Ppa-daf-6(PPA15978). Ppa-C06E8.5 was identified as one of several genes whose transcript was upregulated in the Ppa-obi-1 mutant and is a paralog of Ppa-obi-1 that encodes for a cholesterol-ester binding protein. We found that loss-of-function mutations in Ppa-C06E8.5 do not suppress Ppa-obi-1 hypersensitivity to the host ZTDO, yet they reduce the mutants' ability to recover from dauer stage. Additionally, in C. elegans, DAF-6 has been shown to regulate the shape of the amphid channel at the posterior end of the head region and is also required for lumen formation (WBPaper00025237). DAF-6 encodes a protein known as Patched-related protein localized in the amphid channel. Using CRISPR/Cas9 and F1 mutation detection by High Resolution Melt (HRM), we found nematodes with a potentially gain-of-function Ppa-daf-6 mutation is embryonic and larvae lethal. We hope to use genetic approaches to determine the function of amphid sheath in chemosensation.

Chou, H. et al. (2017) International Worm Meeting "High-Throughput smFISH Analysis to Measure Natural Genetic Variation at Early Developmental Stages."

Deb, D., Paaby, A, Chou, H.

[International Worm Meeting, 2017]

The development of C. elegans is precise and stereotyped, including patterns of cell division in early embryogenesis. Nevertheless, natural genetic variation in wild-type isolates can cause dramatic differences in phenotype following single-gene perturbations, indicating that different wild-type genotypes harbor functional variation in critical gene networks1. These same wild isolates also show extreme variation in the efficacy of germline RNAi1. What are the genetic, molecular and cellular mechanisms that govern these differences? And how do they evolve when stabilizing selection ensures that phenotypic development remains stable and stereotyped? Here we use single-molecule FISH to quantitatively measure the gene expression at specific locations and time points in early development. By characterizing the temporal and spatial heterogeneities of mRNA transcript numbers in the first few cell divisions, we can connect sub-cellular phenotypes to known variations in early embryonic pathway function and germline RNAi. We use a high-throughput, semi-automated pipeline to acquire precise transcript counts at precisely staged embryos, including implementation of the machine learning spot-counting software Aro2. Despite near-invariant cell division phenotypes, wild isolates show significant differences in transcript abundance for critical embryonic genes. These differences in gene expression do not fully explain differences in embryonic lethality following gene knockdown, as neither wild-type gene expression nor transcript abundance following RNAi correlates perfectly with patterns of embryonic lethality. Notably, we observe significant difference in transcript abundance variance following RNAi among wild-type isolates, suggesting inefficiency of RNAi may be controlled by stochastic thresholds. Currently, we are scaling up the experiments using a microfluidic chip specifically designed for worm embryos in order to test hypotheses with high statistical rigor. References: 1- Wild worm embryogenesis harbors ubiquitous polygenic modifier variation. A.B. Paaby, A.G. White, D.D. Riccardi, K.C. Gunsalus, F. Piano, M.V. Rockman. Elife (2015), p. 4 2- Aro: a machine learning approach to identifying single molecules and estimating classification error in fluorescence microscopy images. A.C. Wu and S.A. Rifkin. BMC Bioinformatics (2015), 16:102

Chute, Christopher et al. (2015) International Worm Meeting "Neural circuits involved in octopamine succinylated ascaroside #9 mediated avoidance responses."

Srinivasan, Jagan, Chute, Christopher

[International Worm Meeting, 2015]

Innate behaviors are mediated by the integration of external stimuli and internal signals. These cues often take the form of small biogenic molecules, and range from exogenous compounds to endogenous neuromodulators. These molecules are known to govern behavior across all domains of life. Although the basic components have been characterized in complex systems, the underlying physiological and neural mechanisms are largely unknown. C. elegans is an ideal organism for studying the relationship between small molecules and behavior due to its ability to survive in an unpredictable habitat. In order to successfully navigate their environment, these nematodes must sense and respond to multiple external cues, including physiological state-dependent signals produced and secreted by conspecifics that communicate important ecological contexts. A novel compound of interest, produced exclusively by starved first larval stage worms, is the neurotransmitter-derived metabolite octopamine-succinylated ascaroside #9 (osas#9) [1]. This molecule elicits an acute avoidance response in conspecifics [1], suggesting the communication of unfavorable environmental conditions from starved L1 worms. The avoidance response can be quantified by observing reversal in the worm's locomotion upon application of a drop of osas#9. A reverse genetic approach revealed the involvement of the bioamine neurotransmitters glutamate, octopamine, and tyramine in the avoidance response. Furthermore, through neuronal ablations, the glutamanergic neuron ASH has been identified as the primary sensory neuron necessary for mediating response to this metabolite. Notably, assaying biosynthetic enzymes tdc-1 and tbh-1, known to be required for tyramine and octopamine biosynthesis [2], indicates that the absence of both neurotransmitters does not display a behavioral defect, but the absence of only the downstream neurotransmitter, octopamine, does. This implies the existence of either an uncharacterized antagonistic relationship between octopamine and tyramine, or of an uncharacterized biosynthetic salvage pathway. Since tyramine and octopamine are structurally similar to human catecholamines, understanding their role in governing fear-like states in C. elegans will provide insights on the role of bioamines in mediating human behavior.1. Artyukhin, A.B., et al. J Biol Chem, 2013. 288(26): p. 18778-83.2. Alkema, M.J., et al. Neuron, 2005. 46(2): p. 247-60.

Jha, Sweta et al. (2021) International Worm Meeting "UPS modulation and autophagy."

Jha, Sweta, Holmberg, Carina

[International Worm Meeting, 2021]

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Autophagy is responsible for degrading defective cellular organelles and long-lived proteins in the cytosol and is involved in cell growth, survival, development and death. UPS mostly degrades soluble and short-lived proteins in the nucleus and cytosol and affects various cellular processes. These two proteolytic systems are essential components of the cellular protein quality control system. Several studies have reported an interplay between the UPS and ALP, however it still remains largely unknown how these systems communicate in a multicellular organism. We have recently shown that downregulation of autophagy genes elicits tissue-specific effects on UPS function in C. elegans1. Here, we address how genetic or pharmacological modulation of the proteasome and the proteasome-associated DUBs affects autophagy. We use transgenic C. elegans expressing the autophagy reporters GFP::LGG-12 or mCherry::GFP::LGG-13 and analyze the accumulation of LGG-1 puncta and autophagic flux. Our preliminary data show that downregulation of the proteasome-associated DUBs ubh-4, usp-14 and rpn-11 as well as some proteasomal subunits affect the number of puncta positive for autophagosomes in hypodermal seam cells, intestinal cells as well as in pharynx. To nvestigate a conserved function of the UPS on autophagy in human cells, we are utilizing a HeLa cell line expressing the fluorescence probe GFP::LC3::RFP::LC3G4. A better understanding of the multilayered crosstalk between UPS and ALP in vivo may facilitate development of therapeutic options for various disorders linked to dysfunction in proteostasis. References Jha, S., Holmberg, C.I. Tissue-Specific Impact of Autophagy Genes on the Ubiquitin-Proteasome System in C. elegans. Cells 2020, 9, 1858. Melendez A, Talloczy Z, Seaman M, Eskelinen E-L, Hall DH, Levine B. Autophagy genes are essential for dauer develop-ment and life-span extension in C. elegans. Science 2003. 301:1387-91 Chang J.T., Kumsta C., Hellman A.B., Adams L.M., Hansen M. (2017). Spatiotemporal regulation of autophagy during caenorhabditis elegans aging. Elife. pii: e18459 Kaizuka T., Morishita H., Hama Y., Tsukamoto S., Matsui T., Toyota Y., Kodama A., Ishihara T., Mizushima T., Mizushima N. (2016). An autophagic flux probe that releases an internal control. Mol Cell. 64(4):835-849.

Alan M Zahler et al. (2003) International Worm Meeting "Sup-39 is a U1 snRNA gene which acts as an informational suppressor of a splice donor mutation."

A Brock Roller, John D Tuttle, Alan M Zahler

[International Worm Meeting, 2003]

sup-39 mutations are dominant allele-specific suppressors of the uncoordination defect of unc-73(e936) in C. elegans (1). The unc-73(e936) allele suppressed by sup-39 is a point mutation in the splice donor for intron 16 which changes the canonical G at the first nucleotide of the intron to a T. Given the allele-specific suppression of a splice site defect, we set out to determine whether sup-39 mutations alter the splicing of e936 mutant pre-mRNAs. The e936 mutation activates the use of three different cryptic 5 splice sites, two of which lead to a change in reading frame. The third cryptic site, found in 10% of the stable messages, is unusual; the wild type 5 splice site is used even though it defines an intron beginning with a T. In the presence of a sup-39 mutation, the same three e936 cryptic sites are used, but the levels of the different sites in stable mRNA change; the wild type site represents 35% of stable e936 messages and a concomitant increase in full length UNC-73 protein is detected. We also detect this same effect of sup-39 on chimeric fusion protein constructs. Our initial characterization of the effects of sup-39 on e936 cryptic splicing has been published (2). We have recently cloned sup-39 by SNP mapping. sup-39 is a U1 snRNA gene, one of 12 in the C. elegans genome. Both known alleles possess the same mutation, which is a compensatory mutation in the 5 end of this U1 snRNA to match the mutant splice donor in e936. This is the first example of an informational suppressor (allele-specific gene non-specific suppressor) identified in an snRNA gene by forward genetics. We propose that this U1 snRNA mutation increases the spliceosomes affinity for the mutant 5 splice site. The pre-mRNA sequence requirements for splicing of introns beginning with T will be discussed. We are currently mapping syr-1, a recessive synthetic roller isolated by Jeff Ways group which rolls only in the presence of sup-39 mutations. We hope that identification of a second gene regulated by sup-39 will yield additional clues about U1 snRNA function in recognition of unusual 5 splice sites. 1. Run, J. Q. et al. (1996). Genetics 143: 225-36; 2. Roller, A.B. et al. (2000) Genetics 154: 1169-79.

Tawe WN et al. (1997) Worm Breeder's Gazette "Paraquat mediates differential gene expression in C. elegans"

Henkle-Duehrsen K, Tawe WN, Eschbach M-L, Walter RD

[Worm Breeder's Gazette, 1997]

In order to identify genes that are differentially expressed as a consequence of stress due to paraquat, we used the differential display technique (1) to compare mRNA expression patterns in Caenorhabditis elegans. A C. elegans mixed-stage worm population, as well as a homogeneous larval population were treated with 100 mM paraquat, parallel to controls. Over 50 mRNA species that are potentially up-regulated in response to paraquat were identified. Sixteen of these candidates were re-amplified, ligated into plasmid vectors and the nucleotide sequences were determined. The induction of four of these expressed sequence tags (ESTs) designated L1, M47, M96 and M132 were confirmed in two independent stress / differential display experiments, as well as by northern blot analysis with RNA from stressed and unstressed worms. The nucleotide sequences of the independently isolated L1 and M47 ESTs were found to be identical. Their corresponding mRNA level increased more than 40-fold in the larval stage, and to a lesser extent in the mixed-stage worm population, in response to paraquat. Induction levels of 3 - 5 fold were observed for the M132 and M96 ESTs, in both larval and mixed populations. All of the isolated ESTs showed homology to portions of the C. elegans cosmids. The L1/M47 EST is derived from a gene encoding one of the putative C. elegans glutathione S-transferases. The paraquat-inducible M132 EST was also identified, and encodes a putative C2H2 - type zinc finger protein, possessing an N-terminal leucine zipper. However, the M96 EST appears to be a novel gene whose product has not yet been identified. Searches of other eukaryotic nucleotide sequence databases did not reveal any significant homologies to known sequences from any organism. Since paraquat is known to generate superoxide radicals in vivo, and the cellular superoxide dismutase (SOD) enzyme complex is responsible for the quenching of the deleterious effects of these radicals, the response of the C. elegans superoxide dismutases (2,3,4) to paraquat was also investigated in this study. Northern blot experiments demonstrated that mRNA steady state levels of the C. elegans manganese type and the copper/zinc type superoxide dismutases increased two-fold in response to paraquat, in the larval population. In contrast, mixed-stage populations did not show any apparent increase in the levels of these SOD mRNAs in response to paraquat. References: 1. Liang, P., Pardee, A.B. (1992). Science 257: 967 - 971 2. Giglio, M-P., Hunter, T., Bannister, J.V., Bannister, W.H., Hunter, G.J. (1994). Biochem. Mol. Biol. Int. 33 (1): 37-40 3. Giglio, A. M., Hunter, T., Bannister, J.V., Bannister, W.H., Hunter, G.J. (1994) . Biochem. Mol. Biol. Int. 33 (1): 37-40 4. Larsen, P.L. (1993). Proc. Natl. Acad. Sci. USA, 90: 8905-8909