Yingqi Teng et al. (2001) International C. elegans Meeting "The Roles of cis-elements in the Regulation of C. elegans Hox Gene egl-5"

Scott W Emmons, Yingqi Teng, Henrique B Ferreira

[International C. elegans Meeting, 2001]

Hox genes are well-known segmental identity genes that are conserved in multicellular organisms. Despite the extensive studies on the roles they play during development, how signaling pathways coordinate to activate Hox genes in the proper regions of an organism is not well described. The C. elegans Hox gene egl-5 is involved in many cell fate specification events in the posterior body region. In the seam cell V6 lineage, the expression of egl-5 is under sexual, spatial and temporal regulation. The activation of egl-5 in the seam is male-specific, the expression of egl-5 is restricted to a subgroup of seam cells that give rise to ray 3, 4, 5 and 6, and the earliest activation event always happens at the V6.ppp stage. Another Hox gene, mab-5 , is required for the activation of egl-5 . However, mab-5 is present all through the V6 lineage in both hermaphrodites and males. Therefore the sexual, spatial and temporal specificity of egl-5 expression in the V6 lineage remains unaccounted for. To understand the complicated expression pattern of egl-5 and the convergence of signaling pathways upon activation of egl-5 in a specific lineage, we have been doing PCR-based promoter dissections of egl-5 . A comparison of the intergenic regions upstream of egl-5 in C. elegans and C. briggsae has revealed that there are short stretches of DNA perfectly conserved within the generally diversified non-coding regions. We presume these to be the candidate enhancers of the egl-5 gene. We have been trying to assign them to specific lineages by introducing deletions of these conserved sequences into an egl-5::gfp reporter and looking for changes in the expression pattern. So far, we have roughly mapped the enhancers of rectal epithelial cells and male tail hypodermal cells. Moreover, in a tissue-specific rescue experiment, we have been able to rescue the tail retraction defect in egl-5 mutant males with a construct expressed in the male tail hypodermal cells but not in the rectal epithelial cells or the seam cells. We have also identified a 200bp region highly conserved between C. elegans and C. briggsae as the putative V6 lineage enhancer. This region is not only necessary for the V6 lineage expression of egl-5 , but also gives the wild type expression pattern of egl-5 in the V6 lineage when placed before basal pes-10::gfp. We have found putative binding sites within this conserved region for several relevant transcription factors, namely TRA-1, POP-1 (TCF/LEF1 homolog), and MAB-5/CEH-20 (Extradenticle homolog). Now we are in the process of making smaller deletions and point mutations within the putative V6 lineage enhancer, to identify the functionally significant motifs. Surprisingly, a small deletion of 19bp that contains a putative TRA-1 binding motif lead to ectopic expression of the V6 enhancer driven pes-10::gfp in anterior seam cells, ventral cord and head region in both hermaphrodites and males. The temporal regulation of egl-5 is also disrupted. The same deletion in a full-length egl-5::gfp reporter also causes ectopic expression in the same groups of cells, although to a lesser extent. We hypothesize that this deletion has revealed the binding site of a widely expressed repressor. We are now trying to determine if it is TRA-1 that acts as the repressor. Meanwhile, we are taking a candidate gene approach, looking for loss or ectopic expression of egl-5 in RNAi experiments with known Hox gene regulators such as trithorax related genes.

Michelle S Teng et al. (2005) International Worm Meeting "Reprogramming olfactory behavioural response in C elegans"

Andy Fraser, John McCafferty, Nigel Carter, Bee Ling Ng, Michelle S Teng, Gert Jansen

[International Worm Meeting, 2005]

Odour detection in animals is achieved by virtue of a large family of Olfactory Receptors (OR) expressed in the olfactory epithelium. In humans, the repertoire of odorant receptor genes consists of 1000 genes, each of which encodes a G Protein Coupled Receptor or GPCR. About half of human ORs are pseudogenes. C elegans has 1000 putative OR genes, of which 30% are pseudogenes. Functional expression of ORs in C elegans provides a valuable and novel research tool to efficiently screen for olfactory ligands as well as to study receptor ligand interaction at mid-throughput using chemotaxis response as a quantitative measure. Rat I7 has been functionally expressed in AWA and AWB neurons, which generate attraction and avoidance responses respectively (Milani et al. 2002), resulting in a response to an altered response to octanal and other ligands. We have replicated and extended the initial published observations using a wider range of ligands. Using MoFlo, we have shown that it is possible to separate live transgenic worms co-expressing elt-2 GFP marker from the non-expressors as well as sort them at various stages of the life cycle based on size prior to using the adult animals for chemotaxis assay. We have also extended AWA/AWB targeted expression to another mammalian OR- hOR17-4, which has previously shown to be expressed in both olfactory epithelium and human spermatozoa where it appears to play a role in chemosensory signalling pathways and sperm chemotaxis (Parmentier et al. 1992; Vanderhaeghen et al. 1993). hOR17-4 has been shown to respond to bourgeonal and lilial in HEK293 cells expressing hOR17-4 and in in vitro sperm chemotaxis assays. We were able to reproduce this observation in a simple chemotaxis assay by expressing hOR17-4 driven by odr-10 promoter that targets expression in the AWA neuron. Our preliminary results show a dose dependent migration towards bourgeonal in these transgenic strains at similar concentrations to that published by Spehr et al. (2004).

Michelle Teng et al. (2007) International Worm Meeting "Expression of mammalian GPCRs in C. elegans generates novel behavioural responses to human ligands."

Martijn Dekkers, Michelle Teng, Bee Ling Ng, Suzanne Rademakers, Andy Fraser, Gert Jansen, John McCafferty

[International Worm Meeting, 2007]

G-protein-coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However, purification of GPCRs for biochemical study is difficult and current methods of studying receptor-ligand interactions involve in vitro systems. Caenorhabditis elegans is a soil-dwelling, bacteria-feeding nematode that uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds, making this an ideal system for studying in vivo GPCR-ligand interactions. We sought to test this by functionally expressing two medically important mammalian GPCRs, somatostatin receptor 2 (Sstr2) and chemokine receptor 5 (CCR5) in the gustatory neurons of C. elegans. Expression of Sstr2 and CCR5 in gustatory neurons allow C. elegans to specifically detect and respond to somatostatin and MIP-1alpha respectively in a robust avoidance assay. We demonstrate that mammalian heterologous GPCRs can signal via different endogenous G subunits in C. elegans, depending on which cells it is expressed in. Furthermore, pre-exposure of GPCR transgenic animals to its ligand leads to receptor desensitisation and behavioural adaptation to subsequent ligand exposure, providing further evidence of integration of the mammalian GPCRs into the C. elegans sensory signalling machinery. In structure-function studies using a panel of somatostatin-14 analogues, we identified key residues involved in the interaction of somatostatin-14 with Sstr2. Our results illustrate a remarkable evolutionary plasticity in interactions between mammalian GPCRs and C. elegans signalling machinery, spanning 800 million years of evolution. This in vivo system, which imparts novel avoidance behaviour on C. elegans, thus provides a simple means of studying and screening interaction of GPCRs with extracellular agonists, antagonists and intracellular binding partners.

Fouad, A. D. et al. (2019) International Worm Meeting "Development of a worm picking robot."

Du, A., Li, Z., Fang-Yen, C., Kassouni, A., Fouad, A. D., Teng, C., Bhirgoo, P. D.

[International Worm Meeting, 2019]

C. elegans' small size and manipulability make it an ideal candidate for automation technologies. Numerous automated methods have been developed for handling, imaging, and sorting worms using microfluidics and flow cells. Common to these methods is a requirement that the worms be handled in liquid, which requires special preparation and limits the phenotypes that can be selected for. To our knowledge, no automated method exists to handle worms on agar plates normally used for C. elegans. Here, we describe a worm picking robot capable of transferring worms on standard worm plates using methods similar to those used for manual worm picking. The robot consists of a motorized 3D gantry positioned above a tray of agar plates. The gantry contains a camera to view the plates and a motorized pick than can be actuated linearly or rotationally. Custom software moves the gantry above a requested plate, removes the lid using a vacuum interface, identifies worms on the plate, and then gently lowers the bacteria-coated pick to the worm by capacitive detection of contact between the pick and the agar surface. To sterilize the pick before and after use, a heating coil is extended to cover the pick tip and activated. We demonstrate that our system can identify and autonomously pick L4 and adult animals and safely transfer them to different plates. We developed a scripting language that allows us to coordinate multistep procedures, such as identifying and transferring worms with a specific behavioral, morphological, or fluorescence phenotypes, and setting up genetic crosses. By automating most worm pushing operations, the system will both increase the productivity of researchers during routine experiments and enable experiments that would be impractical using conventional methods.

Michelle S. Teng et al. (2006) European Worm Meeting "Expression of Mammalian GPCRs in C. elegans Generates Novel Behavioural Responses to Human Ligands"

John McCafferty, Martijn P.J. Dekkers, Andrew G. Fraser, Michelle S. Teng, Bee Ling Ng, Gert Jansen, Suzanne Rademakers

[European Worm Meeting, 2006]

Michelle S. Teng1, Martijn P.J. Dekkers2, Bee Ling Ng1, Suzanne Rademakers2, Gert Jansen2, Andrew G. Fraser1 & John McCafferty1. G protein coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However purification of GPCRs for biochemical study is difficult and most methods of screening receptor-ligand interactions require cultured cells and endotoxin free compounds. In contrast, Caenorhabditis elegans is a soil dwelling nematode that feeds on bacteria and uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds. Here we report that expression of the mammalian somatostatin receptor (Sstr2) and chemokine receptor 5 (CCR5) in gustatory neurons allow C. elegans to specifically detect and respond to human somatostatin and MIP-1? respectively in a simple avoidance assay. The endogenous signalling components involved in this remarkable promiscuity of interaction, spanning 800 million years of evolution, are investigated. This system has practical utility in ligand screening. Using structure:function studies, we identified key amino acid residues involved in the interaction of somatostatin with its receptor. This in vivo system, which imparts novel avoidance behaviour on C. elegans, can therefore be used in screening impure GPCR ligands, including the identification of bacterial clones expressing agonists within recombinant libraries.

Yingqi Teng et al. (2002) East Coast Worm Meeting "Understanding the regulation of C. elegans Hox Gene egl-5 through dissection of cis-regulatory elements"

Yingqi Teng, Scott W Emmons

[East Coast Worm Meeting, 2002]

Hox genes are key transcription factors involved in patterning along the anterior-posterior body axis. Although the functions of Hox genes are generally conserved throughout the animal kingdom, in many cases the regulation of Hox genes is not well understood. We are using the male tail system to study the regulation of C.elegans Hox gene egl-5. In the seam cell V6 lineage, egl-5is expressed in a subgroup of precursor cells that give rise to sensory rays 3, 4, 5 and 6. This expression is male specific, restricted to the posterior branches of V6 lineage, and always starts at the V6.ppp stage. We are interested in finding out how the signals that account for the sexual, spatial, temporal and lineage specificity coordinate to activate egl-5 expression in the V6 lineage.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Fouad, A. D. et al. (2019) International Worm Meeting "Automated high-throughput analysis of C. elegans aging using the Worm Collective Activity Monitoring Platform (WormCamp)."

De La Torre, M., Liu, A., De Abreu, C., Xu, J., Hayden, J., Teng, C., Patankar, S., Fang-Yen, C., Churgin, M. A., Fouad, A. D.

[International Worm Meeting, 2019]

C. elegans is a powerful model for investigating the biology of aging. Traditional methods for observing worm aging are slow and dependent on manual observation and manipulation. Our group previously developed an automated multi-well platform (WorMotel) to assay longitudinal C. elegans aging phenotypes of individually isolated worms. Although the WorMotel greatly reduced the amount of manual labor needed to perform aging experiments, scaling of experiments to thousands of conditions for genetic or compound screen is difficult due to the need for manual fabrication of devices and relatively small (240) number of animals per plate. To address these limitations, we developed a complementary method, the Worm Collective Activity Monitoring Platform (WormCamp), which assays aging by monitoring the decline of collective activity of C. elegans populations cultured in standard 24-well plates. To assay the worm populations' aging characteristics, we define a metric based on time required for the cumulative activity distribution for each well to reach a certain fraction of the total cumulative activity. Using validation data derived from WorMotel experiments, we show that this metric provides accurate estimates of lifespan and healthspan. To scale to a large number of conditions, we developed a custom robotic imaging system and software capable of real-time monitoring of lifespan and healthspan in thousands of populations simultaneously. The system consists of a camera and illuminators mounted on a 3D motorized stage positioned above an array of about 100 multi-well plates. The system serially records image sequences from each plate, illuminating it briefly with blue light to stimulate worm activity. We are using the automated system to conduct a whole-genome RNAi screen for genetic interventions that cause changes in lifespan and/or healthspan. The WormCamp method is complementary to the WorMotel method. Since it operates at a population level, the WormCamp provides information on aging with much less detail compared to the WorMotel, but is higher in throughput due to the larger number of animals per plate and the use of standard 24-well plates.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.