Matthew Buechner et al. (2007) International Worm Meeting "Genetic and electrophysiological characterization of the nematode SK3 potassium channel."

Matthew Buechner, Xiangyan Tong, Steve Hoynowski, Kathy Mitchell, Matthew Leming

[International Worm Meeting, 2007]

SK channels are a family of potassium channels with six transmembrane domains and are gated by intracellular Ca2+ ions independent of voltage. Four genes, SK1, SK2, SK3 and IK1, encode the family of SK channels in vertebrates. They are found throughout the nervous system, in smooth muscle, and in epithelial tissues of liver, pancreas, and kidney. Mammalian SK channels are sensitive to the honeybee venom apamin, and have small unitary conductance. We cloned the cDNA for the C. elegans</I> homologue of the SK3 channel, C03F11.1. RACE revealed that the gene surprisingly contains 7 exons upstream of the predicted start site, doubling the size of the predicted protein. The C-terminal half of the protein shows good homology to the mammalian gene, but the N-terminal half is novel. A promoter-gfp construct is expressed strongly in amphid neurons, ventral cord motorneurons, and tail ganglia. Western blots of the worm protein suggest that it is normally bound to calmodulin, as are mammalian SK channels. Patch-clamp analysis of C03F11.1 expressed in CHO cells shows a calcium current similar to that reported for mammalian SK3. We also found that apamin inhibits the conductance of these channels, similar to its effects on mammalian SK3. tm1731</I> animals, created by the Mitani laboratory, are homozygous viable and appear to move normally. We are examining these animals more closely to determine if deletion of C03F11.1 causes subtle defects in motility or behaviour, and using RNAi to determine if deletion of more than one SK channel homologue has synergistic effects on behaviour.

Mitchell PH et al. (2008) European Worm Meeting "Modelling alcohol dependence in Caenorhabditis elegans"

James C, Mitchell PH, Mould RN, Hopper NA, Glautier SP, Dillon J, Oconnor VM, Holden-Dye LM,, Adrianakis I

[European Worm Meeting, 2008]

Ethanol is one of the most widely used and socially acceptable drugs in the. world. However its chronic use can lead to serious problems due to the. development of dependence. Ethanol has been shown have wide-ranging but. selective effects on many different neurotransmitter circuits including;. glutamatergic, GABAergic, serotonergic, dopaminergic and opioid peptidergic. systems. The basis for alcohol tolerance and withdrawal involves. counteradaptive neurochemical changes within these systems to adapt to the. prolonged presence of ethanol in the brain. Here we are using C. elegans to. enable an analysis at all levels of organization from gene, molecule, and. neurone through to neuronal circuit and behaviour. We want to use C.. elegans to investigate the development of neuroadaptation to ethanol. In. order to achieve this we have first provided evidence that ethanol rapidly. equilibriates across the cuticle of C. elegans (Mitchell et al. 2007) thus. enabling experiments in which the concentration-dependent effects of. ethanol on behaviour can be defined. In line with previous studies (Davies. et al. 2003) we have established that external concentrations of ethanol. >100mM are required to overtly affect the visually observable measures of. behaviour. This is equivalent to concentrations that are supra-intoxicating. in mammals. In order to determine whether ethanol elicits any effects on. C. elegans at concentrations equivalent to those that are intoxicating in. mammals we have performed more discreet analyses of behaviours. Pharyngeal. recordings have revealed that concentrations as low as 10mM ethanol have. significant effects on the activity of neural networks, thus providing an. opportunity to investigate the molecular determinants of acute ethanol. effects. We are also in the process of refining the analysis of locomotory. behaviour with the aim of determining whether ethanol elicits effects at. low mM concentrations. As part of this effort we have developed a paradigm. in which we can demonstrate withdrawal from ethanol, and relief from. withdrawal due to low concentrations of acute ethanol. We have used this to. study the onset and recovery rates of this neuroadaptation and the. concentration dependence of this effect in order to understand the. mechanisms involved. This has been extended to investigate if genes already. implicated in acute effects of ethanol in C. elegans are involved in. controlling the withdrawal and tolerance behaviours. These studies are. aimed at better defining how well C. elegans can model aspects of. alcoholism.. Funded by the BBSRC, UK.. References. Davies, A. G., Pierce-Shimomura, J. T., Kim, H., VanHoven, M. K., Thiele,. T. R., Bonci, A., Bargmann, C. I., & McIntire, S. L. 2003, "A central role. of the BK potassium channel in behavioral responses to ethanol in C.. elegans", Cell, vol. 115, no. 6, pp. 655-666.. Mitchell, P. H., Bull, K., Glautier, S., Hopper, N. A., Holden-Dye, L., &. O''connor, V. 2007, "The concentration-dependent effects of ethanol on. Caenorhabditis elegans behaviour", Pharmacogenomics.J., vol. 7, no. 6, pp.. 411-417.

Caroline Bowen et al. (2006) European Worm Meeting "RNA Degradation in the Nematode Worm C.elegans"

Caroline Bowen, Alison Woollard

[European Worm Meeting, 2006]

Caroline Bowen and Alison Woollard RNA turnover is necessary to maintain basal levels of proteins, through mRNA regulation, and to remove aberrant mRNA transcripts. Here we examine processes of RNA degradation in C.elegans. This approach is based on a functional investigation of the C.elegans components of the RNA degradation pathways, identified from homology to other organisms.. The 5-3 RNA degradation pathway in C.elegans has been previously identified (Newbury and Woollard 2004). Here we investigate the 3-5 pathway in C.elegans, starting with the identification of its core and accessory components. 3-5 exoribonucleic acid decay, first identified in S.cerevisae, is brought about by a complex of exoribonucleases, helicases and associated factors, known as the exosome, (Mitchell et al 1997). The exosome was hailed as a proteosome for RNA, due to the degradation properties of the heteromeric complex.. We have used RNAi and an available mutant to show that disruption of the exosome complex, through the targeted destruction of individual components, results in worms with severe developmental problems including slowed growth rates. A full length GFP tagged exosome component, has shown the component to be largely confined to the nucleus of all cells, including the germline. Phenotypes associated with depletion of the exosome components include slow growth and developmental problems. These are attributed to effects on rRNA processing. When exosome components are silenced, there is a failure of the degradation of specific cleaved rRNA precursors, which in turn effects ribosome function and subsequently protein synthesis. We will present our findings on the nature of these rRNA processing defects, which will help to clarify the molecular basis of the phenotypes we observe.

Cunningham K A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "The role of AMP-activated protein kinase in C. elegans feeding regulation"

Ashrafi K, Cunningham K A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The evolutionarily conserved AMP-activated protein kinase (AMPK) is a critical energy sensor in eukaryotes. In general, upregulation of AMPK leads to shutting down energy consuming processes and activation of energy generating processes. In C. elegans, the catalytic alpha subunit, encoded by aak-2, has been shown to have roles in regulating fat storage during the hibernating dauer state as well as longevity in both dauer and non-dauer animals.1,2 We found that aak-2 deficient animals have increased feeding rate relative to wild type animals, and reconstitution studies suggest that expression in pharyngeal muscle and the nervous system is sufficient for restoration of wild type feeding rate to aak-2 mutant animals. Moreover, using various neuron specific promoters, we found that neural sufficiency of aak-2 for feeding regulation can be restricted to the small subset of neurons defined by an hlh-34 promoter. Interestingly, hlh-34 encodes for a basic helix-loop-helix transcription factor whose mammalian counterpart, sim-1, has known developmental and regulatory roles in specific regions of the hypothalamus. Mammals deficient in SIM-1 develop obesity and exhibit increased feeding behavior.3 Thus, the role of AAK-2 in hlh-34/sim-1 expressing cells in modulating metabolism and feeding behavior may be conserved across evolution. References: 1 Narbonne, P. and Roy R., 2009. Nature. 457, 210-213. 2 Apfeld, J., O'Connor, G, McDonagh, T., DiStefano, P.S., Curtis, R., 2004. Genes and Dev. 18, 3004-3009. 3 Michaud, J.L., Boucher, F., Melnyk A., Gauthier, F., Goshu, E., Levy, E., Mitchell, G.A., Himms-Hagen, J., Fan, C.M., 2001. Hum. Mol. Genet. 10, 1465-73.

O'Connor V et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Modelling neuroadpative plasticity underpinning ethanol withdrawal in C. elegans"

Mould R, Holden-Dye L, O'Connor V, Mitchell P, Dillon J, James C

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Ethanol the major pharmaco-active component alcoholic drinks has a range of effects that arise over a tight dose response curve. Increasing concentrations (20-200 mM) of ethanol are associated with distinct behavioural states encompassing intoxication, anaesthesia and ultimately death (as neural activity that drives respiration is inhibited). In the case of chronic ethanol consumption humans exhibit a distinct behavioural state when ethanol is withdrawn and this is typically associated with tremor and anxiety. C. elegans has previously been used to define mechanisms and molecules underpinning the acute effects of ethanol (Davis &amp; McIntire, 2004, Biol Proc Online 6, 113-9). We have taken advantage of our observation that the cuticle does not represent a barrier to ethanol entry (Mitchell et al., 2007 Pharmacogenomics, 7. 411-7), to define a dose and time dependent paradigm in which ethanol conditioned worms can be driven into a withdrawn state upon ethanol removal. This state is assayed by measuring worms locomotory behaviour. Motility of population of worms to a food source or by individually monitoring single worms by video analysis defines dysfunction relative to untreated controls. Further this shows that the disruption is distinct to that associated with acute intoxication. A striking feature of the withdrawn state is the large increase in number of omega turns relative to untreated controls. This increase is also associated with the majority of the omega turns being independent of a reversal. In humans, low doses of ethanol provide relief from withdrawal and represent an important component of the negative reinforcement associated with alcoholism. We can model this as low doses of ethanol that otherwise do not effect locomotory behaviour relieve the disruption in locomotion seen by ethanol withdrawal. We have parameterized worm locomotion to define a score of normal and ethanol disrupted behaviour. By screening several mutants, against this criteria identified several neuromodulatory transmitters, including monoamines and neuropeptides, which are essential in induction and/or maintenance of the withdrawn state. In view of the pivotal role these transmitter pathways play in the neuronal plasticity that underpins alcohol dependent behaviours in humans, the withdrawal paradigms in C. elegans could provide a platform for a gene to behaviour analysis of the homeostatic mechanism that control alcoholism.

Yu, Xinwei et al. (2021) International Worm Meeting "Fast deep learning correspondence for neuron tracking and identification in C. elegans using semi-synthetic training"

Sharma, Anuj K., Linderman, Scott W., Randi, Francesco, Yu, Xinwei, Leifer, Andrew M., Creamer, Matthew S.

[International Worm Meeting, 2021]

The nervous system of the nematode C.elegans is well characterized, such that each of the 302 neurons is named and has stereotyped locations across animals(Witvliet et al., 2020). The capability to find corresponding neurons across animals is essential to investigate neural coding and neural dynamics across animals. Despite the worm's overall stereotypy, the variability in neurons' spatial arrangement is sufficient to make predicting neural correspondence a challenge. We present an automated method to track and identify neurons in C. elegans, called "fast Deep Learning Correspondence" or fDLC, based on the transformer network architecture(Vaswani et al., 2017). The transformer has shown great success in natural language processing tasks by modeling the dependencies between words in a sentence. We reasoned this same architecture would be well-suited to extract spatial relationships between neurons in order to build a representation that facilitates finding correspondence to neurons in a template worm. The model is trained once on empirically derived semi-synthetic data and then predicts neural correspondence across held-out real animals via transfer learning. The same pre-trained model both tracks neurons across time and identifies corresponding neurons across individuals. Performance is evaluated against hand-annotated datasets, including NeuroPAL(Yemini et al., 2020). Using only position information, the method achieves 80.0% accuracy at tracking neurons within an individual and 65.8% accuracy at identifying neurons across individuals. Accuracy is even higher on a published dataset(Chaudhary et al., 2021). Accuracy reaches 76.5% when using color information from NeuroPAL. Unlike previous methods, fDLC does not require straightening or transforming the animal into a canonical coordinate system. The method is fast and predicts correspondence in 10 ms making it suitable for future real-time applications. Witvliet D, Mulcahy B, Mitchell JK, Meirovitch Y, Berger DR, Wu Y, et al.Connectomes across development reveal principles of brain maturation in C.elegans. bioRxiv. 2020;doi:10.1101/2020.04.30.066209. Vaswani A, Shazeer N, Parmar N, Uszkoreit J, Jones L, Gomez AN, et al.Attention is All You Need; 2017.Available from:https://arxiv.org/pdf/1706.03762.pdf. Yemini E, Lin A, Nejatbakhsh A, Varol E, Sun R, Mena GE, et al. NeuroPAL: A multicolor atlas for whole-brain neuronal identification in C. Elegans. Cell.2020;doi:10.1016/j.cell.2020.12.012. Chaudhary S, Lee SA, Li Y, Patel DS, Lu H. Graphical-model framework for automated annotation of cell identities in dense cellular images. eLife.2021;10:e60321. doi:10.7554/eLife.60321.

Waterston RH (1986) Worm Breeder's Gazette "a candidate myo-3 hypomorphic allele."

Waterston RH

[Worm Breeder's Gazette, 1986]

The gene myo-3 which codes for the minor body wall myosin heavy chain myoA (Miller et al PNAS in press), has been localized to LGV near Sma-1 (Albertson, EMBO 4, 2493-2498). Increased accumulation of myoA through tandem duplication of the gene suppresses unc-54(0) mutations, but hypomorphic or null alleles of the gene have not been identified. One postulate has been that myo-3(0) mutations when homozygous are lethal. To look for lethal mutations that might lie in the myo-3 gene, Kathy Kellerman, a rotation student at Washington University, and I screened over 700 mutagenized F1 clones of a sma- 1/daf-11 strain (see map below) for clones failing to produce expected numbers of normal Sma-1 progeny. Of the several mutations identified, one, st378, resulted In the arrest of homozygous animals as paralyzed L1's. The st378 mutation has several properties consistent with the hypothesis that it is a myo-3 hypomorphic allele as described below, but the relatively small numbers of purified homozygotes obtained so far have frustrated efforts to measure myoA levels directly. The st378 mutation is closely linked to, and right of, Sma-1 as shown on the map below, based on 2 and 3 factor data. Although the Sma-1 mutation has been separated from st378, a chromosome carrying only gt378 has not been obtained yet. The Sma-1(e30) st378 homozygotes proceed normally through embryonic cell divisions and resemble Sma-1 in morphogenesis, with the exception that the double mutant never changes its position achieved at the end of elongation. Only slight movements of the tip of the head and tail are visible. Hatching occurs only slightly later than the wild type sibs. The pharynx pumps and bacterial ghosts are present in the L1 gut, yet the animal arrests in the L1 stage without ever unfolding. ctDf1/e30 st378 animals have a similar if not identical phenotype. Electron microscopy of Sma-1(e30) st378 homozygotes show that the body wall myofilament lattice is abnormal. Few normal thick filaments are present; in addition there are filaments larger than the normal thick filaments of irregular outline. The st378 is essentially recessive in a wild type background, but is semi dominant in unc-56(e190) or unc-15(e73) backgrounds. The unc-54( e190); ) st378/++ animals for example are more severely paraiyzed than unc-54(e190) homozygotes, and the few animals of this genotype that reproduce have very small brood sizes. sup-3 mutations in trans counterbalance these effects; e.g. unc-54(e190); 07)/sma-1(e30) st378 animals resemble unc-54(e190) homozygotes, and are more paralyzed than unc-54(e190); 07)t+ animals. The ability in the nematode to identify a protein of interest, to recover its DNA and to localize it by cytogenetics, and to obtain subsequently mutations in the genomic copy of that gene is an approach of considerable potential.

P. Mitchell et al. (2006) European Worm Meeting "Evidence that Ethanol Equilibriates Rapidly Across C. elegans Cuticle"

L. Holden-Dye, N.A. Hopper, K. Bull, V. O'Connor, P. Mitchell, S. Glautier

[European Worm Meeting, 2006]

P. Mitchell, K. Bull, N.A. Hopper, S. Glautier1, L. Holden-Dye, V. O''Connor Ethanol has concentration-dependent effects on neural function. At concentrations in the low mM range, equivalent to the drink-driving limit, it has an intoxicating action, which is often associated with a transient excitability, followed by inhibition. At higher concentrations, greater than 100 mM, it has an anesthetic action and elicits muscle paralysis. C. elegans has been used to define the genetic basis for behavioural responses to ethanol exposure. In these studies intact animals have been exposed to 500 mM ethanol with the prediction that the internal concentration is more than ten-fold lower and therefore equivalent to an intoxicating, rather than an anesthetic, dose (Davies and McIntire, 2004). In order to underpin further studies, we have analysed the relationship between the external and internal ethanol concentration of C. elegans. Immersion of C. elegans in a sealed chamber containing ethanol (range 100 mM to 500 mM) elicits an inhibition of thrashing rate. Inhibition reaches a steady-state value within 5 min. This effect is concentration-dependent (half-maximal effect at 300 mM). At all concentrations of ethanol a steady-state inhibition is observed within 5 min. The animals rapidly recovered when removed from ethanol. Notably, this recovery was complete within 2.5 min. This suggests that ethanol may rapidly equilibriate across the cuticle and that the internal concentration that is required to inhibit motility approximates to the external concentration. In order to directly assay the effects of ethanol on muscle we used the pharyngeal muscle preparation. In this assay the muscle is exposed and therefore the concentration of ethanol is known. This muscle is inhibited by ethanol at concentrations in excess of 100 mM. Although it could be argued that the pharyngeal muscle behaves in a different fashion to body wall muscle in terms of ethanol sensitivity, the results support the contention that concentrations in excess of 100 mM are required to inhibit muscle activity. Internal ethanol concentrations in C. elegans following exposure to ethanol have been estimated using a biochemical assay kit. However, this procedure requires that the animals are briefly washed in cold buffer prior to the measurement. Our observation that animals exposed to ethanol fully recover from the inhibition of motility within 2.5 min suggests that a significant amount of ethanol may be lost from the inside of the animals during the protocol. We tested this by determining the influence of the wash volume on the apparent estimate in internal ethanol concentration. This shows that the estimate of internal ethanol concentration increases as the volume of the wash buffer decreases. This suggests that the measurement of internal ethanol concentration using this method underestimates the true internal concentration.. Davies, A.G. and McIntire, S.L. 2004, Biol. Proced. Online 6, 113-119.. Funded by the BBSRC, UK

Dhillon I et al. (2018) MicroPubl Biol "GABA Receptor mutant gbb-1 accelerates morphological aging of GABA neurons in Caenorhabditis elegans"

Dhillon I, Chin-Sang I, Momin A

[MicroPubl Biol, 2018]

The aging of an organism is heterogeneous, that is to say, tissues and organs may age at unique rates compared to overall organism longevity. Inhibition of the insulin/insulin-like growth factor signaling (IIS), such as the daf-2 IIS receptor increases Caenorhabditis elegans longevity. The daf-2 pathway plays a key role in the regulation of metabolism and stress responses and as such, impacts the longevity of the organism (Uno and Nishida 2016). Evidence suggests that an increase in insulin signaling results in a reduction in overall lifespan and a decrease in neuronal longevity (Kenyon 2010). Recently, the gbb-1 GABA signaling pathway has also been implicated in the regulation of adult longevity (Chun et al. 2015). The GABAergic motor neurons innervate the dorsal and ventral body muscles controlling locomotion, foraging and defecation of the animal. GABA, an inhibitory neurotransmitter, has been found to decrease the lifespan of the nematodes (Chun et al. 2015), however, its role in neuronal aging has yet to be determined. The formation of ectopic neurite branches and production of axonal beads are two known phenotypes of neuronal aging in C. elegans. In this study, we examined the aging of the GABAergic motor neuron nerve cords and dorsal commissures. Fifty age-synchronized worms were imaged every three days (days 3, 6, 9, 12, 15, 18). Worms were plated on Fluorodeoxyuridine (FUdR) treated agar plates in order to maintain only adult worms throughout the imaging process (Mitchell et al. 1979). Neurons were visible using the GABAergic specific unc-25::GFP marker strain. To keep the data consistent, images were taken of the GABAergic axons and dorsal commissures between the VD5 and DD5 cell bodies (Panel A, scale bar 10 microns). Fluorescent images were taken using the Zeiss Axioplan fluorescent microscope under 40x objective. To prepare slides for fluorescence microscopy, an agarose immobilization protocol was followed (Fang-Yen et al. 2009). Aging of the neurons was quantified by counting the number of axonal beads and branches present on the neural processes. Branched processes emanating from the cell body, neural axon or dorsal commissures were scored as aging phenotypes. As predicted, GABAergic neurons displayed phenotypic evidence of aging. The C. elegans genome encodes two metabotropic GABAB receptor genes, gbb-1 and gbb-2, which are homologous to their mammalian counterparts (Chun et al. 2015). GABA binds to the gbb-1 receptor initiating a signaling cascade that ultimately inhibits the daf-16 transcription factor. A reduction of function mutation in the gbb-1 encoded GABA receptor subunit increases worm lifespan; however, it seems to be specific to the GBB-1 subunit as no effect was observed in a loss of function gbb-2 mutation. When overexpressed, gbb-1 mutants were short-lived which is consistent with GABA signaling regulating lifespan (Chun et al. 2015). To determine the effect of GABA signaling on neuronal aging, we observed the effect of a loss-of-function gbb-1 mutant on the aging of the GABAergic motor neurons. Unexpectedly, the GABAergic dorsal commissures and axons in the gbb-1 mutants displayed a significant increase in aging morphology compared to the wild type (Panel A). gbb-1 mutants displayed a significant increase in beading (Panel B, Table 1) and branching (Panel C, Table 2) in the GABAergic motor neurons compared to the wild type from day 9 onwards (p<0.05, independent t-test, n=50). Thus gbb-1(tm1406) mutants although long lived, have neurons that appear to age faster. This work was done with a single gbb-1 allele, in the future it will be important to verify our findings with additional gbb-1 alleles or show that a transgene with the wildtype gbb-1 can suppress the observed aging phenotypes.

Edgar RS et al. (1977) Worm Breeder's Gazette "the nematode C elegans: a new organism for intensive biological study."

Wood WB, Edgar RS

[Worm Breeder's Gazette, 1977]

A small free-living soil nematode is receiving close scrutiny from a growing number of biological researchers. Some of these investigators believe that Caenorhabditis me the E. coli or at least the bacteriophage T4 of the animal world. C. elegans is a small transparent worm about a millimeter in length. Its genes are carried on five autosomes and a sex chromosome (X), and it has a genome size about 20 times that of E. coli. It generally reproduces as a self-fertilizing hermaphrodite (XX), but occasional males (XO), which arise by nondisjunction, permit sexual reproduction as well. The worm's prime virtues for research are its short life cycle (3 days) , ease of cultivation on E. coli as a food source, simple anatomy ( 810 somatic nuclei) and convenience for genetic analysis. Building upon the pioneering work of Dougherty (1), Nigon (2), and especially Brenner (3), who first described the animal's genetics, there are now more than a dozen laboratories in Canada, France, Germany, India, Japan, the United Kingdom, and the United States that are investing heavily in studies of this organism. From April 13 to April 16, researchers from these laboratories gathered for the first time to assess the status of their investment at a conference in Woods Hole, Massachusetts, supported in part by a grant from the National Institute of Aging, NIH. Many investigators initially turned to C. elegans in the hope of understanding its behavior in terms of its simple nervous system. The status of this work was a major topic at the meeting. C. elegans contains only 256 neurons, and a complete anatomical description of the nervous system at the electron microscope level is virtually at hand. Previous work had allowed detailed reconstruction of the head anterior sensory neuroanatomy, the ventral nerve cord, and the associated dorsal cord composed of processes from cells in the ventral cord (4). At the meeting, John White (MRC Cambridge) described reconstruction of the complex nerve ring that surrounds the pharynx and presumably plays the major role in processing sensory inputs to produce motor outputs. D. Hall (Albert Einstein College of Medicine) presented a reconstruction of the tail ganglia carried out in R. Russell's laboratory at Caltech. As a result of these studies, a complete wiring diagram for the whole nervous system of the animal is now within reach. Due to its small size, the system's function cannot yet be approached directly by standard electrophysiological techniques, and must await technical advances in electrode technology or in optical methods using potential-sensitive dyes. Meanwhile, however, Tony Stretton (University of Wisconsin) has shown that the anatomy of the related nematode Ascaris, which is sufficiently large for conventional electrophysiological studies, is virtually identical to that of C. elegans at least in the ventral cord. John Walrond from Stretton's laboratory presented evidence on which of the seven types of motor neurons in the ventral cord are inhibitory and which stimulatory. As a taste of things to come, R. Russell (now at University of Pittsburgh) presented a model to account for control of movement in C. elegans based on its ventral cord circuitry. More complete and detailed nervous system models should become possible as more functional features of the anatomy become understood. Another approach to obtaining such functional information is the investigation in several laboratories of nematode neurotransmitters. Earlier work by J. Sulston (MRC, Cambridge) localized three catecholamine-containing neurons, but mutants that did not produce catecholamines showed no demonstrable alteration in behavior (5). C. Johnson (University of Wisconsin) reported that in Ascaris acetylcholine appears to function as an excitatory transmitter, on grounds that it is synthesized in excitatory but not in inhibitory motor neurons. Johnson also reported on work carried out in R. Russell's laboratory at Caltech on the identification of a C. elegans mutant defective in acetylcholinesterase and another defective in choline acetyltransferase (the latter isolated in the laboratory of D. Hirsh, University of Colorado, as resistant to the drug trichlorofon). Jim Lewis (Columbia University) reported on the properties of putative acetylcholine receptor mutants isolated as resistant to the acetylcholine analog levamisole. R. Horvitz (MRC, Cambridge) reported on the behavior of mutants that fail to accumulate serotonin normally in certain neurons. The combination of biochemical, genetic, anatomical, and physiological approaches being taken by these researchers seems certain to provide insight into the processing of neuronal signals in C. elegans, and eventually could lead to an understanding of the animal's behavior at the level of its neuroanatomy and neurophysiology. Recently many investigators have recognized the potential of C. elegans for the study of development. The major portion of the meeting was devoted to description of the worm's development at the cellular level, and to studies of mutations that perturb it. The adult animal has only 810 somatic nuclei in six major cell types ( hypodermis, muscle, neurons, gut, gonadal sheath, and coelomocytes), and its anatomy at the cellular level is virtually invariant. A current major effort in the descriptive work has been the determination of cell lineages, and an important result of the meeting was the prospect that the complete cell lineage of C. elegans soon will be established from the zygote to the adult animal. Upon hatching from the egg, the juvenile or first-stage larva has only 540 somatic cells. During larval development, about 200 post- embryonic cells arise from division of a few blast cells present at hatching. J. Sulston and R. Horvitz (MRC, Cambridge) had previously traced in detail the origins of these post-embryonic cells (6). Their lineage studies revealed a remarkable reproducibility from one animal to another in times of cell division, paths of cell migrations, cell deaths, and ultimate differentiated cell fates. In experiments reminiscent of Boveri's classical observations on Ascaris embryogenesis, E. Schierenberg and other workers in G. von Ehrenstein's laboratory (Gottingen) now have established cell lineage in the egg up to the 186-cell embryo. Their studies, carried out with the light microscope using differential interference contrast optics and time lapse video recording reveal that characteristic rates of cell division are maintained in different subclones, regardless of cell migrations. The Gottingen group also has made detailed reconstructions of a 294-cell and 540-cell embryo from electron micrographs of serial sections. Surprisingly, the 540-cell stage is reached quite early in embryogenesis, about midway between fertilization and hatching and prior to most of the cell growth and differentiation that takes place before hatching, indicating that cell division and migration precede differentiation per se. Nevertheless, from the correspondence between the positions of cells in the 540-cell embryo and first stage larva, von Ehrenstein has been able to identify most of the embryonic cells with regard to their future fates. There is optimism that the remaining gap, between the 186-cell and the 540- cell stages can be filled by a combination of light and electron microscopy, to provide for the first time a complete description at the cellular level of the development of an animal from the egg to adulthood. The post-embryonic lineage of the gonad somatic cells, not investigated by Sulston and Horvitz, now has been determined by J. Kimble (University of Colorado). The gonad arises during larval development from a 4-celled precursor structure in both the hermaphrodite and the male. In the hermaphrodite the two somatic cells of the precursor give rise to a total of 142 cells, which form the gonadal sheath, spermatheca, and uterus. In the male a similar lineage produces about 50 cells to form the spermatheca and vas deferens. As in other tissues, the lineages are invariant in their significant features. The lineage in another single organ, the intestine, has been followed by K. Lew working in S. Ward's laboratory (Harvard Medical School), taking advantage of a mutant discovered by P. Babu (Tata Institute, Bombay; 7) in which gut cells fluoresce due to a defect in tryptophan catacolism. Lew has traced the lineage of the gut cells, which begin to fluoresce during embryogenesis, from a single precursor cell in the 8-cell embryo through the 20-cell juvenile gut to the adult 32-cell gut. He also has found aberrations in the lineage pattern in certain embryonic lethal mutants. On a related project, P. Siddiqui in P. Babu's laboratory reported that X irradiation of embryos heterozygous for such a fluorescence mutation gave rise to adults with fluorescent patches in the gut, suggesting an approach to the study of somatic crossing over in C. elegans.The factors responsible for lineage patterns are being investigated using a laser to selectively destroy certain cells, or using mutations that alter lineage. The preliminary results so far reported suggest that whereas there are some inductive or positional effects of cell differentiation, much of the developmental process is cell-autonomous. Two examples of nonautonomy were reported by J. White (MRC, Cambridge). Neuroblast cells that in a particular mutant fail to migrate to the normal position fail to differentiate completely into nerve cells. Also, if gonad development is prevented by laser ablation of the precursor cells, then the hypodermal cells that normally proliferate to form the vulva fail to do so, By contrast, other studies revealed a remarkable degree of cell autonomy. D. Albertson (MRC, Cambridge) reported on the behavior of certain blast cells that normally undergo a migration followed by a pattern of division to form 6 neurons and a hypodermal cell. In a particular mutant strain the blast cells undergo up to three abortive attempts at division to yield a polyploid cell, which differentiates to display cellular features of both hypodermal and nerve cells. R. Horvitz (MRC, Cambridge) reported on current progress in the genetic dissection of lineage patterns. He has isolated a large number of mutants that are defective in vulva formation and hence cannot lay eggs. These mutants so far define 15 different genes in which defects appear to directly affect the vulval lineage pattern, by preventing either divisions or normal migrations of precursor cells. Two laboratories are investigating differentiation of sperm, which are amoeboid cells in C. elegans. A large number of temperature- sensitive sperm-defective mutants have been isolated by D. Hirsh ( University of Colorado; 8), and S. Ward (Harvard Medical School), and analysis of these mutants is proceeding. At non-permissive temperature, some mutants appear to be blocked early in spermatogenesis and form no sperm, whereas others produce normal numbers of inactive sperm. All the mutants can lay viable eggs when mated with normal males. Ward reported that male sperm migrate rapidly to the hermaphrodite spermatheca following copulation and effectively supplant the endogenous sperm. Sperm may be required for some step in oogenesis, because some sperm-defective mutants fail to produce oocytes alone, but will do so when mated to males. The abundance of mutants and the prospects for isolating sperm in quantity make this system promising for studying the genetic control of a cellular differentiation pathway. Development of the male is virtually identical to that of the hermaphrodite until some hours after hatching, when a few of the post- embryonic cell lines display a male-specific pattern of division, migration, and differentiation. Intersex and transformer mutants defective in genes that control these processes were described by M. Klass (University of Colorado), Ward, and J. Brun (Lyon, France). These mutants should prove useful in determining how the male developmental pattern is superimposed upon that of the hermaphrodite. A special feature of the C. elegans life cycle has been studied by D. Riddle (University of Missouri). Normally, the young hatched larva progresses through a series of four molts to adulthood. In the absence of food, however, the second molt produces a special form, the dauer larva, which has an altered cuticle and can withstand adverse conditions (e.g., 4% SDS) and no food for periods up to 60 days. When presented with food, the dauer larva molts and continues the normal progress toward adulthood. Riddle has identified 7 genes whose functions are involved in the choice between the regular and the dauer developmental pathways. Mutants defective in these genes are constitutive dauer formers which enter the dauer pathway even in the presence of food. Some of these mutants have defects in sensory neuronal anatomy. Mutant analysis indicates that a larger number of genes probably is essential for recovery of the dauer larva and return to the standard developmental pathway. Brenner had estimated earlier that C. elegans carries about 2000 genes (3), somewhat fewer than Drosophila. If so, then more than 10% of them already have been identified. R. Horvitz (MRC, Cambridge) has undertaken the task of collating mapping data from different laboratories, and reported that over 200 genes have been identified and mapped with various degrees of precision. This number promises to increase rapidly, because investigators in the laboratories of R. Herman (University of Minnesota), D. Baillie (Simon Fraser University) , and W. Wood (Caltech) are embarking on exhaustive studies of lethals in defined regions of the genome. This work will be aided greatly by the availability of chromosomal duplications, deficiencies, and translocations, some of which already have been isolated and characterized in Herman's laboratory for use as crossover suppressors and balancers for lethals. Herman also reported on the isolation of stable tetraploid strains and on the instability of triploids generated by crosses to diploids. These studies incidentally provide information on the sex-determining mechanism in C. elegans, which as in Drosophila depends upon the balance between the numbers of sex chromosomes and autosomes. Promising studies also were reported on molecular genetic analysis of genes for muscle proteins. A large number of mutants defective in myosin, paramyosin (Q protein of the thick filaments) and other muscle components previously had been reported by R. Waterston and H. Epstein while working in Brenner's laboratory (9). At the meeting, Epstein (Stanford University) reported on the anatomical and biochemical properties of C. elegans muscle. At least 6 genes have been identified as being involved in muscle development. Of these, 2 appear to be the structural genes for myosin and paramyosin. Epstein reported that at least 2 different myosins exist in the nematode, one in the body wall muscle and another in the pharynx. The identified myosin gene controls the structure of the body wall muscle myosin. Some 30 alleles of this gene have been found, including a small internal deletion. S. MacCleod (MRC, Cambridge) reported on biochemical mapping of the mutational alterations in defective myosins by chemical cleavage and peptide analysis. Myosin and paramyosin mutations also have provided possible genetic access to the translational apparatus of C. elegans. R. Waterston ( Washington University) has shown that revertants of certain myosin mutations carry a second site suppressor that suppresses chain- terminating mutations in the myosin and paramyosin genes and certain alleles in genes with other diverse functions. This suppressor mutation is a promising candidate for the first tRNA nonsense suppressor to be described in a metazoan organism. C. elegans also is well suited to studies on the phenomenon of aging, in view of its short life cycle and the existence of a non- aging developmental variant, the dauer larva (10). M. Klass ( University of Colorado) reported on the effects of nutrition and other parameters on life span, and D. Mitchell (Boston Research Institute) presented evidence for entrainment of increased life span by prolonged cultivation under semi-starved conditions. However, attempts to find mutations that directly alter the life span so far have failed, and progress in this general area has been minimal. In a panel discussion following the session on aging the participants agreed that C. elegans has many potential advantages as an aging model, but that fruitful approaches exploiting genetics have yet to be developed. Many other studies were presented in addition to those mentioned. In this brief summary we have attempted only to highlight what in our opinions were the major themes of a rich and exciting meeting. The group of investigators who gathered at Woods Hole is taking a somewhat unusual holistic and cooperative approach to understanding the biology of a single organism. The meeting was invaluable in bringing together senior researchers and students with backgrounds in molecular, cellular, developmental, and neurobiology, in fostering the spirit of cooperative and integrated inquiry, and in generating a mutual enthusiasm that will help to make C. elegans an important model organism for intensive biological study during the next few years.