Vera Lahl et al. (2001) International C. elegans Meeting "Early embryogenesis differs even in close relatives of C. elegans"

Vera Lahl, Marzena Kutzowitz, Christian Halama, Stefan Ingensand, Einhard Schierenberg, Magdalena Laugsch

[International C. elegans Meeting, 2001]

We have started to study close and more distant relatives of C. elegans for their pattern of embryogenesis. While many differ only marginally from C. elegans, others show considerable differences. These include the sequence of divisions, variations in the spatial arrangement of blastomeres, presence or absence of polarity reversal in the germline, requirement of early zygotic transcription for cleavage, mode of cell specification, gastrulation, and temperature tolerance. Variations in many but not all variations can be understood as different aspects of heterochrony. We find that,depending on the parameter considered, species closer related to C. elegans may nevertheless express stronger differences than species phylogenetically more distant. This suggests that environmental factors influence the pattern of early development.

Stefan Junemann et al. (2006) European Worm Meeting "Deciphering the Role of Serotonin Blocking Muscle Degeneration"

Laurent Segalat, Stefan Junemann

[European Worm Meeting, 2006]

Stefan Jnemann and Laurent Sgalat. Duchenne muscular dystrophy (DMD) is a progressive myopathy which is caused by the disruption of the dystrophin gene. Until today, there is no effective cure of the disease. Neither do we understand completely the function of the dystrophin protein. In C. elegans, homologues for dystrophin and most of its associated proteins have been identified. In addition, a dys - 1/CeMyoD double mutation displays an activity - dependent, progressive degeneration of muscle fibres resembling the DMD. We therefore use C. elegans as a model system to get insights in both the function of dystrophin and in possible treatments of DMD. In a screen of biochemical compounds in the dys - 1/CeMyoD-/- genetic background, the neurotransmitter Serotonin was found to reduce significantly the degeneration of muscles. In order to decipher the pathway involved in this muscle - specific Serotonin function, we performed an EMS mutagenesis to screen for supressor mutants of the Serotonin effect in the dys 1/CeMyoD background. Of 5000 F1 animals tested in our assay, we obtained 13 candidates which reduce the Serotonin effect to different degrees and at different stages, respectively. In particular, the Serotonin induced protection from muscle cell disorganisation, which apparently precedes cell degeneration and that is characterized by the loss of nuclei and perturbation of mitochondria pattern, is blocked in almost all supressor mutants examined. Future work will reveal the identity of the mutated genes and hopefully shed more light on the role of Serotonin in muscle function.

Stefan Eimer et al. (2006) European Worm Meeting "The Integral Membrane Protein UNC-50 is required for Nicotinic Receptor Trafficking in C. elegans"

Janet Richmond, Alexander Gottschalk, Michael Hengartner, Stefan Eimer, Jean-Louis BESSEREAU, William R Schafer

[European Worm Meeting, 2006]

Stefan Eimer1, Alexander Gottschalk2, Janet Richmond3, Michael Hengartner4, William R Schafer2, and Jean-Louis Bessereau1. Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that undergo multiple assembly, modification and transport steps prior to insertion into the plasma membrane. However, little is known about the molecules that are specifically participating in these processes. In C. elegans a nAChR sensitive to the nematode-specific cholinergic agonist levamisole has been well characterised. Mutants in all of its four subunits confer resistance to muscle hyper-contraction and death upon exposure to levamisole.. Similarly, unc-50 mutant animals exhibit the same resistance to paralysis and death upon exposure to levamisole. By antibody staining and electro-physiological recording of body-wall muscles we show that levamisole receptors are not detectable at cell surface in unc-50 mutants. However, a second type of muscle nAChR, which is insensitive to levamisole, and the GABA receptor are normally transported to the synapse. unc50 encodes an integral membrane protein conserved from yeast to man. In C. elegans, unc-50 is ubiquitously expressed and localized to the Golgi system. We showed that UNC-50 is required in body wall muscles for the transport of the assembled levamisole receptor to neuromuscular junction. In the absence of UNC-50 the receptor is sorted within Golgi to the lysosomal system and rapidly degraded. Therefore, UNC-50 might be part of/constitute a Golgi resident check point during levamisole receptor transport. UNC-50 interacts with the GBF1 class of large Arf GEFs which are involved in retrograde transport within the Golgi system. Models how retrograde transport and sorting would required for levamisole receptor trafficking will be discussed.

Avery L et al. (2000) West Coast Worm Meeting "A Muscarinic Contribution to the Regulation of Feeding"

Avery L, Steger KA

[West Coast Worm Meeting, 2000]

Acetylcholine (Ach) is essential for worms' survival and can stimulate feeding through nicotinic receptors on the pharyngeal muscle. However, we suspect that Ach affects the pharynx through muscarinic receptors as well. Worms that lack Ach (e.g. cha-1 mutants) have a more severe feeding defect than worms that lack pharyngeal nicotinic transmission (eat-2; eat-18). Atropine, a muscarinic antagonist, causes worms to appear starved, although it increases their pumping rate. Three ORFs in the worm genome closely resemble vertebrate muscarinic receptors: C15B12.5 (acm-1), F47D12.2 (acm-2) and C53A5.12 (acm-3). We have examined the expression of these three genes: all three are expressed in pharyngeal tissue (muscle, neurons or both) and in the extra-pharyngeal nerve ring. We have obtained mutant alleles of two putative muscarinic receptors: acm-1, and acm-2 (a gift from Stefan Eimer and Rolf Baumeister). The two mutations have very different effects on worm physiology. acm-2 worms are hypersensitive to aldicarb (an inhibitor of cholinesterase) and to nicotine. They pump more rapidly than wild type worms in the absence of drug, and decrease their pumping rate in the presence of atropine. We suspect that acm-2 mutants are defective in down-regulating nicotinic transmission. We hypothesize that acm-2, which is expressed in neurons both inside and outside the pharynx, acts as a negative regulator at nicotinic synapses. acm-1 mutants, in contrast, are resistant to aldicarb, and are not hypersensitive to nicotine. In an unc-17 background of reduced cholinergic transmission, acm-1 worms are hypersensitive to atropine. We suspect that acm-1, probably in combination with acm-3, affects pumping efficiency or the adaptation of feeding behavior to particular circumstances. We are currently designing assays and appropriate genetic backgrounds to examine these possibilities

Thomas Boulin et al. (2006) European Worm Meeting "Adaptation to the Nicotinic Agonist Levamisole"

Jean-Louis BESSEREAU, Stefan Eimer, Janet Richmond, Thomas Boulin

[European Worm Meeting, 2006]

Thomas Boulin1, Stefan Eimer1, Janet Richmond2, Jean-Louis Bessereau1. Chronic exposure to receptor agonists often leads to long lasting changes in synaptic function and adaptation to these drugs. Analysis of the adaptation process can provide interesting paradigms to get further insights into mechanisms of synaptic plasticity. We are using a genetic approach to characterize the adaptation behavior to levamisole, a nematode-specific nicotinic agonist. In C. elegans, body-wall muscles are innervated by inhibitory GABAergic and excitatory cholinergic motoneurons. Fast excitatory transmission is mediated by nicotinic acetylcholine receptors. Levamisole activates acetylcholine receptors at neuromuscular junctions and causes paralysis and death of wild-type worms after acute exposure to high concentrations (1mM). However, when wild-type animals are chronically exposed to sublethal concentrations of levamisole (0.1mM), they still enter paralysis but ''adapt'' and regain partial mobility after 6-12 hours. Adapted worms are uncoordinated and resemble mutants of levamisole-sensitive AChR subunits. Indeed, adapted worms gain resistance to 1 mM levamisole, suggesting that responsiveness to levamisole is reduced in muscle. Consistently, levamisole-specific currents recorded during bath application of levamisole are reduced by 50% in adapted worms. This effect is specific since the response to GABA remains unchanged in adapted animals. The reduction of levamisole currents could be explained by a number of scenarios. First, receptor levels could be diminished. Immunofluorescence staining of UNC29 and UNC-38, two subunits of the levamisole- sensitive AChRs, indicates that receptors remain visible and clustered after adaptation. Precise quantification of fluorescence signals are in progress to detect potential variations of receptor amounts at synapses. Second, subcellular redistribution of the levamisole receptor via endocytic mechanisms could be involved in adaptation. Indeed, immunofluorescence staining does not allow us to differentiate between receptor in the plasma membrane or in adjacent sub-cellular compartments. Finally, the quality of receptor present in the muscle membrane could be changed. For instance, post-translational modifications of the receptor subunits could modulate the sensitivity to levamisole.. In order to identify the molecular mechanisms involved in adaptation, we are testing candidate mutants affecting various physiological processes. So far, we have shown that the calcium-dependent protein-phosphatase calcineurin/tax-6 is required in body wall muscle for adaptation to levamisole.

Ruvkun GB et al. (2000) East Coast Worm Meeting "Genetic analysis of neural inputs to the dauer pathway"

Ruvkun GB, Sandoval GM

[East Coast Worm Meeting, 2000]

C. elegans uses sensory information about its environment to decide whether to proceed with development or arrest at the dauer larval stage. Signalling through converging TGF-b/DAF-7 and insulin-like/DAF-2 neuroendocrine pathways is required for non-dauer development. Animals mutant in daf-7, a TGF- b super family member (Ren at al. Science 274, 1996), arrest development inappropriately at the dauer larval stage. A study to identify the neurotransmitters used in the neural inputs into the dauer pathways revealed that daf-7 animals can return to normal reproductive development by the application of the acetylcholine muscarinic receptor agonist oxotremorine (Tissenbaum et al. PNAS, 2000). Muscarinic agonists do not induce dauer recovery in animals defective for an insulin-like receptor DAF-2 which are daf-c. This suggests a role for muscarinic signalling in an insulin-like pathway. We have taken two approaches to further investigate the role of muscarinic signalling in the dauer pathway. We have identified three potential muscarinic receptor homologues (acm-1, acm-2 and acm-3) by sequence comparison in the C. elegans genome sequence database. We have analysed the expression of a GFP promoter fusion of each of these three genes. All constructs are expressed in a small set of neurons with one construct also exhibiting expression in intestinal cells. The pharynx also exhibits differential expression with acm-2 showing expression in the pharyngeal neurons and acm-3 in the pharyngeal muscle cells. Currently, deletion mutants of acm-1 (from Kate Steger, Leon Avery) and acm-2 (from Stefan Eimer, Ralf Baumeister ) are being analysed for dauer phenotypes and genetic interactions with genes in the dauer pathway. To further address the role of acetylcholine signalling in the dauer pathway we made double mutants between daf-c genes and animals with mutations that have decreased cholinergic signalling. We observed an L1 arrest in the double mutant of the insulin-like receptor daf-2 (e1370) and a vesicular transporter of acetylcholine unc-17 (e245) (Alfonso, A. et al. Science 261, 1993) but not with daf-7(e1372); unc-17(e245) . To isolate other components in this signalling pathway we have done a screen to suppress this L1 arrest and isolated daf-2 and unc-17 suppressors. We are in the process of characterising and mapping these mutants.

Kate Steger et al. (2001) International C. elegans Meeting "ACM-2, a neuronal muscarinic acetylcholine receptor, contributes to the regulation of feeding"

Leon Avery, Kate Steger

[International C. elegans Meeting, 2001]

The pharynx of C. elegans is a neuromuscular pump, whose coordinated contraction and relaxation allows the worm to efficiently ingest bacteria. In wild type worms, pharyngeal muscle contraction is triggered by the motor neuron MC, which releases acetylcholine to nicotinic receptors on the pharyngeal muscle. Pharyngeal pumping can respond to changes in the worm's surroundings, suggesting additional regulatory influences on pharyngeal muscle. Muscarinic acetycholine receptors are likely to play a role in such regulation; three genes that appear to encode muscarinic receptors are expressed in pharyngeal tissue, and investigations into the G o / G q signaling network have revealed a muscarinic component. acm-2 encodes a G-protein coupled muscarinic acetylcholine receptor that is expressed in neurons of the extra-pharyngeal nerve ring and in pharyngeal neurons I1, I2 and M4. A deletion mutant, acm-2(by124) , was generated by Stefan Eimer and Ralf Baumeister (Ludwig-Maximilians-Universitaet, Munich). We have previously reported that acm-2(by124) confers a feeding phenotype: pharynxes of mutant worms display an increased pumping rate and an increased sensitivity to nicotine. We had also shown that gap junctions are necessary for the full expression of the mutant phenotypes. A gap junction links the extra-pharyngeal neuron RIP to the pharyngeal neuron I1, and this connection may be important for ACM-2 action. However, gap junctions also link neurons within the pharynx. We have since determined that the pumping phenotypes of acm-2(by124) can be rescued by the injection of wild type copies of the acm-2 genomic region. We have also noted that the phenotype of increased nicotine sensitivity is dominant. Because the by124 mutation removes two exons without inducing a frame shift or premature stop, we hypothesize that acm-2(by124) encodes an antimorph. We have continued to examine the effects of the by124 mutation, and found additional effects on worm behavior. The mutation alters the worm's ability to respond to different types of food: while wild type worms increase their pump rate when presented with certain types of bacteria, acm-2(by124) worms do not. We hypothesize that ACM-2 acts in a pathway that allows environmental cues to influence pharyngeal pumping rate. We suspect that MC is the ultimate target of this pathway, and that information from the extra-pharyngeal nervous system alters pumping rate by adjusting the frequency of MC firing. ACM-2 is an inhibitory element of the pathway, perhaps providing negative feedback at stimulatory cholinergic synapses. We have also found that acm-2(by124) mutants change direction while crawling more often than do wild type worms. This observation suggests that ACM-2 plays a similar negative regulatory role in several behavioral pathways. We are currently attempting to identify the site of action of ACM-2 within each behavioral pathway, and to further characterize the dominant nature of the by124 mutation.