Martijn Dekkers et al. (2006) European Worm Meeting "Dissecting Gustatory Plasticity in C. elegans Using Mammalian Receptors"

Martijn Dekkers, Gert Jansen, John McCafferty, Michelle Teng

[European Worm Meeting, 2006]

Martijn Dekkers1, Michelle Teng2, John McCafferty, Gert Jansen1. We use C. elegans to study the molecular and cellular mechanisms of salt perception, using behavioural assays and calcium imaging. We discriminate three distinct responses to NaCl: First, attraction to NaCl concentrations ranging from 0.1 to 200 mM. Second, avoidance of higher concentrations. Third, avoidance of an otherwise attractive NaCl concentration after prolonged exposure. We call this latter behaviour gustatory plasticity. Previous studies have shown that chemo attraction to NaCl is mediated primarily by ASE, and to a lesser extent by ASI, ADF and ASG, and avoidance of high concentrations of NaCl is mediated by ASH (Bargmann & Horvitz, 1991). In our lab we have identified 85 proteins and five pairs of gustatory neurons that mediate gustatory plasticity. Based on our results we propose a model in which prolonged exposure to 100 mM of NaCl, elicits a signal from the ASE neurons, leading to sensitisation of the avoidance signalling ASI, ADF, ADL and ASH neurons. This results in avoidance of low concentrations of NaCl.. In an effort to identify the roles of the individual cells in gustatory plasticity we expressed either a TRP channel or a G-Protein Coupled Receptor (GPCR) in the neurons that have been implicated in gustatory plasticity. This allows us to specifically activate those cells. The TRP channel that we use is the mammalian capsaicin receptor VR-1. Normally C. elegans does not respond to capsaicin. Previously it has been shown that expression of VR-1 in the ASH neurons results in avoidance of capsaicin (Tobin et al 2002). We have generated animals that express the VR-1 receptor in the ASE, ASI, ADL and ADF neurons. We are currently testing their responses to capsaicin and the effects of preexposure to NaCl on this response, using behavioural assays.. The GPCRs that we have chosen are the mouse SSTR-2 somatostatin receptor and the human CCR-5 chemokine receptor. We have expressed these receptors in the ASH cells, and tested the responses in a novel avoidance assay. We found that the transgenic animals display specific avoidance behaviour to the ligands of the receptors, indicating that these GPCRs are integrated into the endogenous C. elegans signalling machinery, which is remarkable, given the evolutionary distance between the species. We are now making constructs to express these GPCRs in the other cells to assess their role in gustatory plasticity.

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.

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.

Laskova, Valeriya et al. (2013) International Worm Meeting "Mapping the entire connectome of C. elegans L1 larvae."

Zhen, Mei, Kasthuri, Bobby, Shalek, Richard, Lichtman, Jeff, Samuel, Aravi, Pfister, Hanspeter, Laskova, Valeriya, Kaynig-Fittkau, Verena, Wen, Quan, Berger, Daniel, Guan, Asuka

[International Worm Meeting, 2013]

To investigate the poorly understood mechanisms of development and function of the nervous system, connections between neurons must be deciphered first. The adult nervous system of C. elegans was mapped to near completion by Dr. John White and his colleagues in 1970s. However, neuronal wiring in C. elegans larvae differs from that of an adult, since it undergoes multiple rounds of neuronal birth, apoptosis and rewiring during development. We utilize an Automatic Tape-Collecting Ultramicrotome (ATUM) to section an entire L1 stage animal into thousands cross-sections, followed by the automated imaging with a scanning electron microscope (SEM) to map an entire neuronal wiring diagram with synaptic resolution. The acquired wiring diagram will guide and be validated by calcium imaging to map the functional connection of the juvenile motor circuit.

Phuongnhung Nguyen et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Dissecting Genetically the Neural Networks that Coordinate Locomotion"

Bill Walthall, Gennady Cymbalyuk, Phuongnhung Nguyen

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

The networks of nerves and muscles responsible for forward and backward movements in C. elegans offer unique advantages for investigating the functional relationships among cells in integrated cellular networks. The first advantage is that John Sulston and his colleagues identified the interneurons, motor neurons and muscle cells that compose the networks (Sulston, et al., 1983). Second, John White and colleagues determined the synaptic patterns that interconnect the network (White et al., 1986). Finally, the C. elegans community has generated mutations that cause specific alterations in the cellular networks. A model based on the connectivity pattern and laser ablation results suggests that distinct sets of interneurons and excitatory motor neurons are dedicated to forward and backward movement, respectively. These two circuits converge upon two classes of inhibitory motor neurons and four longitudinal strands of body wall muscles and create antiphasic contractile muscular waves that travel along the dorsal and ventral axes of the body. We will report results from the analysis of movies showing locomotion patterns of animals. These movies allows us to quantitatively characterize the traveling wave in terms of amplitudes, dorsal and ventral deflections, frequency and velocity of the wave progression and the forward and backward velocity of the animal's progression. We will compare wild-type locomotion characteristics with those exhibited by three uncoordinated mutants (unc-4, unc-55, and cnd-1) that are known to make specific alterations in the network that result in movement defects that are predicted by the model. The connectivity model can also be used to predict epistatic relationships for double mutant combinations (for example unc-55 mutants exhibit a ventral asymmetric pattern of backward movement, whereas unc-4 exhibits a dorsal asymmetric pattern of backward movement). We find that unc-4 masks the defect of unc-55 mutants, which is predicted by the model neural circuit. However the model's predictions for forward movement are not as accurate. We will discuss possible explanations for the resiliency of forward movement and the vulnerability of backward movement to genetic perturbations.

Consortium Wormbase et al. (2000) West Coast Worm Meeting "From ACeDB to a more complete and usable database"

Consortium Wormbase, Foltz NL, Schwarz EM, Sternberg PW

[West Coast Worm Meeting, 2000]

We briefly describe the current status and plans for WormBase, initially an extension of the existing ACeDB database with a new user interface. The WormBase consortium includes the team that developed ACeDB (Richard Durbin and colleagues at the Sanger Centre; Jean Thierry-Mieg and colleagues at Montpellier); Lincoln Stein and colleagues at Cold Spring Harbor, who developed the current web interface for WormBase; and John Spieth and colleagues at the Genome Sequencing Center at Washington University, who along with the Sanger Centre team, continue to annotate the genomic sequence. The Caltech group will curate new data including cell function in development, behavior and physiology, gene expression at a cellular level, and gene interactions. Data will be extracted from the literature, as well as by community submission. We look forward to providing the C. elegans and broader research community easy access to vast quantities of high quality data on C. elegans. Also, we welcome your suggestions and criticism at any time. WormBase can be accessed at www.wormbase.org.

Ken-ichi Oshio et al. (2002) Japanese Worm Meeting "Revision of synaptic connectivity database"

Eizo Akiyama, Yuishi Iwasaki, Yuko Osana, Ken-ichi Oshio, Sohei Gomi, Satoru Morita, Kotaro Oka, Kazumi Omata

[Japanese Worm Meeting, 2002]

The synaptic connectivity of C. elegans is well known from observations of the somatic system by White et al. and those of the pharyngeal system by Albertson et al. So far, three databases were constructed for computational usage by Achacoso et al. and Durbin, and recently in WormBase. However, they lack some data such as those in tables of White's paper and those in figures of Albertson's book. Our database (K. Oshio, S. Morita, Y. Osana and K. Oka: Technical Report of CCEP, Keio Future No.1, 1998) includes all data described in White's paper and Albertson's book. Unfortunately, some mistakes were found in the database through private communications with John White who is the author of White's paper and with the users of the database. Thus we have been proceeding with the revision to make it perfect one. We are planning to complete the revision in September 2002. The database should be worthwhile not only for neurophysiological studies, but also for post-genomic interests mediating genomes and behavior.

Kevin Eliceiri et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "A Swept-Field Confocal for Cell Biology Studies"

Kevin Eliceiri, Mike Szulczewski

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

We describe a novel instrument, The Swept-Field Confocal Microscope (SFC) that combines high resolution pinhole imaging with slit imaging. Unlike spinning disk confocal systems that have their pinhole apertures embedded in a spinning disc, the SFC's 32 pinhole array remains stationary. Galvonometric and piezo controlled mirrors sweep the image of the pinholes across the sample. The emission photons are de-scanned and focused through a complementary set of pinholes onto a CCD camera. This results in a high resolution image that can be collected at up to 100 frames/second in the pinhole mode and greater than 1000 frames per second in the slit mode. The ability for the scientist to match the optical recording with the temporal biological fluorescence response has great promise for cell and developmental biology studies where live dynamic events must be captured quickly in high resolution. In this poster we describe the optical path of the microscope and present some early data in collaboration with John White's group at the UW-Madison of using the SFC to image GFP constructs in C .elegans to investigate cell division in the early embryo.

Tylon Stephney et al. (2007) International Worm Meeting "WormImage: An online photo album for C. elegans electron microscopy."

Huawei Weng, Maurice Volaski, David H. Hall, Toni Long, Kevin Fisher, Meng Xu, Tylon Stephney

[International Worm Meeting, 2007]

The Hall lab is the home for the physical EM data for C. elegans that was collected by many scientists working at several key laboratories over the past 40 years. We have almost all of the annotated prints, negatives, data notebooks and often the original thin sections and blocks from the MRC, Missouri, Caltech and AECOM collections, including the work of John White, John Sulston, Sam Ward, and many others. This archive includes wild type and mutant data, and covers both males and hermaphrodites, and all ages from embryo to adulthood, and some aging animals. We are scanning much of this physical print data to produce a digital archive of C. elegans anatomy. We have generated about 3 terabytes of digital images, and there is much more scanning to be done. We have designed a simple online photo album, WormImage, to share some of this archive as small thumbnail versions that can quickly transit across the Internet to all users, for free. Bandwidth issues limit our ability to ship full size scans electronically, but we also supply users with higher resolution scans upon request, by ftp or on DVDs. WormImage (www.wormimage.org) is an online database developed for remote retrieval of this digitized microscopy data. Currently this database contains about 20,000 different digital images. Most scans have been taken from the workprints, to preserve original hand annotations which mark identified cell types. The WormImage database is updated weekly to provide more images. The user can search this database by animal name, age, sex, or by regional information (head, midbody, tail) or by tissue types to identify potential images of interest. The user can quickly survey many animals in small thumbnail images, or concentrate on details of a few images expanded to larger size. The program was recently revised to make it easy for the user to click through all the thumbnails for one animal in serial order. Hall has been reviewing the original annotations and writing brief summaries for each animal, also offered on the website. These Color Code summaries help to translate the shorthand markings on the original prints into the familiar cell names for many structures. During 2007, WormImage will begin showing images of key mutant phenotypes and aging animals. We are grateful to Demian Nave and Art Wetzel at the Pittsburgh Supercomputing Center for their help in providing a mirror site. This work is funded by NIH RR 12596.