Queiros, Libania et al. (2021) International Worm Meeting "Evidence for the inclusion of Caenorhabditis elegans in Environmental Risk Assessment routines"

Queiros, Libania, J. M. Goncalves, Fernando, Pereira, Patricia, Pacheco, Mario, Aschner, Michael, L. Pereira, Joana

[International Worm Meeting, 2021]

Caenorhabditis elegans has long been used as a choice for a laboratory model organism, and it has been linked to several discoveries that have led to Nobel Prizes. Although C. elegans' journey as an invaluable model in developmental biology and neurobiology is long, dating to the 1960s, its application in other research fields such as environmental toxicology is more recent. As a well-known experimental organism bearing high sensitivity to different environmental contaminants, and representing important functional levels in soil and aquatic ecosystems, C. elegans has high potential to be extensively integrated within Environmental Risk Assessment (ERA) routines. The major advantages supporting the inclusion of C. elegans in lower tiers of ERA, where a first screening of potential hazardous scenarios to the biota of both environmental compartments is performed, as well as the large array of endpoints that can be tested in this context, are herein presented. In addition, its sensitivity to contaminants such as metals and pesticides, as well as specific strengths and limitations, are compared to other laboratorial model organisms commonly used in ERA, such as Daphnia magna (crustacean) and Eisenia fetida (earthworm). The inclusion of C. elegans in ERA routines is encouraged, since it may provide ecologically relevant insights on the effects of contaminants, thus improving the establishment of appropriate environmental protection benchmarks.

Maria Vidal et al. (2004) Mid-west Worm Meeting "spd-3 is a novel gene required for spindle alignment in C. elegans"

John G White, Maria Vidal

[Mid-west Worm Meeting, 2004]

In order to develop a complex organism, cells must have the ability to divide asymmetrically to produce two daughter cells of different developmental potential. A cell accomplishes this by polarizing cytoplasmic components to opposite ends such that, upon cleavage, each daughter inherits distinct components. This segregation requires coordination of the axis of polarity and the cleavage plane. In animal cells, the cleavage plane is specified by the alignment of the mitotic spindle. To better understand the molecular mechanism of spindle alignment we are studying a temperature sensitive, maternal effect, lethal mutation in the spd-3 gene of C. elegans . Cytological analysis reveals that the spd-3(oj35) mutant is defective in nuclear and spindle positioning. During the first mitosis the spindle fails to align along the anterior/posterior axis leading to abnormal cleavage configurations. spd-3(oj35) mutants also exhibit a failure to extrude polar bodies, which could be attributed to a misaligned meiotic spindle. Post-embryonic defects, including uncoordination and sterility, are consistent with cell division defects indicating that SPD-3 may be involved in spindle alignment in all tissues. The spd-3(oj35) strain contains a mutation converting Leucine130 to Phenylalanine in H34C03.1. The H34C03.1::GFP transgene rescues the spd-3(oj35) phenotype indicating that the novel gene H34C03.1 is spd-3 . Surprisingly, SPD-3::GFP localizes to mitochondria. However, this may not be unprecedented because the kinesin Kip3 is required for microtubule instability and spindle alignment in S. cerevisae (DeZwaan, 1997; Miller, 1998) and its Drosophila homolog localizes to mitochondria (Pereira, 1997). In studying genes such as spd-3 we hope to reach a better understanding of factors involved in spindle alignment which is essential for defining the plane of cytokinesis and ensuring proper inheritance of segregated cytoplasmic components.

Joseph Gallagher et al. (2006) European Worm Meeting "Development and Optimisation of Mos1 for Gene Tagging in C. elegans"

Joseph Gallagher, Patricia Kuwabara

[European Worm Meeting, 2006]

Joseph Gallagher and Patricia Kuwabara. The Drosophila transposon, Mos1, has been used successfully to generate insertional mutants in C. elegans1. Mos1 can be mobilised in C. elegans worms, which carry two independent extrachromosal arrays, one encoding the Mos1 transposase under the control of a heat shock promoter, and the other carrying copies of the Mos1 transposase substrate. Heat induction of the transposase leads to the mobilisation of the transposon and its integration within the C.elegans genome. Stable Mos1 insertions are detected by PCR after animals have been clonally passaged for a number of generations. As a member of the NemaGENETAG Consortium, which is funded through EU fp6, we are working with our partners to optimise the already existing Mos1 tools and to develop new strains to facilitate the high-throughput screening of C. elegans Mos1 insertional mutants. The generation of C. elegans transposon tagged genes will enhance our understanding of gene function, especially those that are associated with human disease, and provide an invaluable resource to the research community.. Here, we present our preliminary results, which were obtained after screening a library consisting of almost 1000 strains. So far, from this and subsequent screens, over 300 Mos1 strains have been identified by PCR. Each of these strains has been frozen at 80?C in triplicate, test thawed and re-tested to validate the presence of a Mos1 insertion. Many of the characterised strains carry multiple copies of Mos1 inserted at different sites, so the actual number of tagged loci is greater than the number of strains frozen. Steps are now being taken to improve the rate of transposition and the recovery of mutants. 1.. Bessereau, J. L. et al. Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature 413, 70-4 (2001).

Loer, C. M. et al. (2019) International Worm Meeting "Comparative neurotransmitters and neuronal specification in the satellite model organism nematode Pristionchus pacificus."

Sommer, R. J., Witte, H., Loer, C. M., Hobert, O.

[International Worm Meeting, 2019]

All bilaterian animals use the same small molecule 'classical' neurotransmitters in their nervous systems. Some basic mechanisms of cell fate specification also seem to be shared among most animals, despite their being separated in evolution by more than 500 million years. Yet, actual nervous system structures and functions do vary greatly among animals; we understand much less about how neurons and nervous systems change during evolution. We are generating tools and strains to study such evolution by comparing the nervous systems of C. elegans (Cel) and other nematode 'satellite model organisms' such as Pristionchus pacificus (Ppa). In C. elegans, the COE-type transcription factor UNC-3 acts as a terminal selector that specifies ventral nerve cord (VNC) cholinergic motorneuron (MN) fate; a chordate ortholog also specifies cholinergic fate in Ciona (Kratsios et al., 2012, Nat Neurosci 15:205). We tagged the endogenous Ppa-unc-3 locus with 2x-FLAG using CRISPR-Cas9, and examined its expression by immunofluorescence. The pattern of UNC-3-positive cells in Pristionchus in the VNC appears identical to that of C. elegans; it is similar but not identical in the head and tail. [Cel UNC-3 specifies cholinergic but not always other fates in many cholinergic head and tail neurons (Pereira et al., 2015, eLife 4:e12432; Kratsios et al., 2015, Curr Biol 25:1282).] We also examined GABA-immunoreactive cells in Pristionchus, and found the pattern identical to C. elegans in the VNC and tail. The pattern of Ppa GABA neurons in the head is similar to that of C. elegans, but still being determined. All Pristionchus VNC neurons can be accounted for by cells expressing UNC-3 (presumptive cholinergic MNs), GABA, or 5HT (4 presumptive VCs). We are continuing to examine neuronal fate expression in Pristionchus and to generate strains useful to these pursuits.

Peter I. Joyce et al. (2006) European Worm Meeting "The Atypical Calpains of C. elegans"

Patricia E. Kuwabara, Peter I. Joyce

[European Worm Meeting, 2006]

Peter I. Joyce and Patricia E. Kuwabara . Regulated proteolysis of receptors, cytoskeletal proteins and transcription factors is an important process that modulates cell growth and development. A family of calcium-regulated thiol proteases, known as calpains, has been shown to perform such processes in mammals, and other eukaryotes. Loss of function mutations in calpains are linked to certain pathological conditions including limb-girdle muscular dystrophy 2A (LGMD2A); whereas, gain of function mutations are associated with the formation of cataracts. . We are investigating the biochemical and functional role of atypical calpains in C. elegans. We have identified seven atypical calpains and eight calpain-like sequences within the C. elegans genome. To gain an understanding of their possible roles in C. elegans development we have created calpain promoter transcriptional expression constructs for the atypical calpains 1 to 7 using monomeric RFP. The transcriptional expression patterns were analysed by co-localisation with the following tissue specific GFP markers: unc-119::GFP (all neurons), unc-47::GFP (GABAergic neurons within the ventral nerve cord), myo-3::GFP (body wall muscle), exc::GFP (excretory cell) and seam::GFP (seam cells). Five out of the seven atypical calpains show promoter transcriptional expression activity; clp-1, clp-2, clp-4, tra-3 and clp-7. Two of the atypical calpain promoter constructs fail to show detectable transcriptional expression activity; clp-3 (lacks all catalytic residues) and clp-6 (possible pseudogene). clp-1 shows the highest expression throughout the animal, specifically in neurons and body wall muscle. . In parallel with expression studies we have taken a global approach into studying calpain function using RNAi, calpain mutant analysis and ectopic over-expression. Preliminary studies have revealed no obvious effects. Because calpains require calcium for proteolytic activity, we are investigating ways in which calcium levels can be perturbed within C. elegans, as a way to activate calpains ectopically. We are also undertaking biochemical studies to characterise the proteolytic properties of the atypical calpains.

Karen A O'Hanlon et al. (2005) International Worm Meeting "Identification and Analysis of Enhanced Life Maintenance Genes in C. elegans."

Ann M Burnell, John A Brown, Lisa M Knox, Karen A O'Hanlon

[International Worm Meeting, 2005]

Mutations in genes of the daf-2 insulin/insulin-like growth factor signaling (IIS) pathway have been found to extend adult lifespan in C. elegans1. This increase in lifespan resulting from decreased IIS is supressed by a loss of function of the daf-16 gene which encodes a forkhead transcription factor2,3. A microarray analysis was carried out at the Sanger Institute on two temperature sensitive (ts) long-lived strains of C. elegans: daf-2(sa193) and daf-2 (m41), and compared their transcription profiles to those of wild type N2 and daf-7 (e1372) ts adult worms. From this study, 1,700 genes were identified as being 2-fold up- or down-regulated in the long-lived nematodes. These 1,700 genes were compared to the gene expression profiles obtained from other microarray ageing studies4,5,6. Based on these comparisons we selected 20 up- and 20 down-regulated genes for gene knockout analysis by RNAi. An RNAi feeding protocol was designed using the ts sterile C. elegans mutant glp-4 (bn2 ts). glp-4 nematodes were fed on the E. coli dsRNA feeding strains and lifespan was measured by counting the number of live nematodes every second day. A long-lived control consisting of a C. elegans glp-4 fed on daf-2 dsRNA expressing E. coli (daf-2 knockout) and a normal lifespan control consisting of C. elegans glp-4 fed on E. coli transformed with the empty vector (no gene knockout) were also used. These experiments are ongoing. Our results to date show that RNAi of 4 down-regulated genes increases lifespan and RNAi of 3 up-regulated genes decreases lifespan. We will report on our progress on these RNAi experiments. We are grateful to Dr. Patricia Kuwabara in whose laboratory the microarry experiment was carried out, to Dr. David Gems and Dr. Joshua McElwee for helpful discussions and to Enterprise Ireland for financial support. 1. Kenyon, C. et al. (1993). Nature 366, 461-464. 2. Lin K. et al. (1997). Science 278, 1319-1322. 3. Ogg. S. et al. (1997). Nature 389, 994-999. 4. Murphy, C.T. et al. (2003). Nature, 424, 277-284. 5. Kimura, K.D. et al. (1997). Nature, 277, 942-946. 6. McElwee J.J. et al. (2004). J. Biol. Chem. 279, 44533-44543.

Huo, J. et al. (2019) International Worm Meeting "Dissecting neural mechanisms for motor sequence generation in C. elegans."

Zhen, M., Wen, Q., Alkema, M., Hung, W., Wang, Y., Zhang, X., Xin, Q., Xu, T., Huo, J.

[International Worm Meeting, 2019]

There are two prevailing views of neural control of sequential activities in the brain. In the first scheme, feedforward excitation in a synaptic chain would transiently activate different groups of neurons1,2. In the second scheme, sequential neural activity could emerge from mutually inhibited neuron clusters through a winner-take-all strategy3. Here, we investigate neural mechanisms for motor sequence generation in C. elegans during escape responses. Upon a mechanosensory or thermosensory stimulus at a worm's head, the animal will move backward, generate an Omega turn, and then move forward in a new direction4. We found that this rudimentary motor sequence is controlled by a neural circuit that combines the above two schemes. In particular, feedforward excitation is contributed by electrical couplings between interneurons AIB, which encode the backward motor state, and RIV in the turning module via innexin proteins such as INX-15,6. Mutual inhibitions are contributed by glutamate synaptic inputs from AIB to neurons encoding turning and forward motor states5; and cholinergic synaptic inputs from neurons in the turning and forward module to those in the backward module through acetylcholine-gated chloride channels (e.g., acc-1, acc-2, acc-4)7. Remarkably, when neurons in the turning module were ablated, the statistics of escape responses became consistent with stochastic transitions between backward and forward attractor states at constant rates, leading to prolonged reversals with an exponential dwell time distribution. In other words, feedback inhibition from the turning module plays a critical role in terminating persistent activity in the backward module. Together, our data suggest that feedforward excitation and selective inhibitions between neuron groups contribute to robust and flexible motor sequence generation in C. elegans. Reference: 1. Abeles, M. Corticonics: Neural Circuits of the Cerebral Cortex. Cambridge University Press. 1991. 2. Long M A, Jin D Z, Fee M S. Support for a synaptic chain model of neuronal sequence generation. Nature, 2010, 468(7322): 394. 3. Seeds A M, Ravbar P, Chung P, et al. A suppression hierarchy among competing motor programs drives sequential grooming in Drosophila. Elife, 2014, 3: e02951. 4. Chalfie, M. et al. The neural circuit for touch sensitivity in Caenorhabditis elegans. J Neurosci 5, 956-964 (1985). 5. White J G, Southgate E, Thomson J N, et al. The structure of the nervous system of the nematode Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci, 1986, 314(1165): 1-340. 6. Altun Z F, et al. High resolution map of Caenorhabditis elegans gap junction proteins. Developmental Dynamics, 2009, 238. 7. Pereira L, et al. A cellular and regulatory map of the cholinergic nervous system of C. elegans. Elife, 2015, 4: e12432.