Mark J Alkema et al. (2003) International Worm Meeting "Tyramine Modulates Foraging Behavior and Head Movements."

Mark J Alkema, Bob Horvitz, Melissa Hunter-Ensor, Niels Ringstad

[International Worm Meeting, 2003]

Tyramine and octopamine are biogenic amines implicated in several invertebrate behaviors. Tyramine biosynthesis requires the decarboxylation of tyrosine. Octopamine is synthesized by subsequent -hydroxylation of tyramine. We obtained mutant strains carrying deletions in a putative C. elegans amino acid decarboxylase gene (tdc-1) (gifts from M. Dong and M. Koelle) and a tyramine--hydroxylase gene (tbh-1). Using HPLC analysis and radioenzymology we showed that both the tdc-1 and tbh-1 deletion mutants lack octopamine, indicating that tdc-1 and tbh-1 are required for octopamine biosynthesis. Furthermore, we showed that TDC-1 can decarboxylate tyrosine in vitro. TDC-1 and TBH-1 expression overlap in the RIC interneurons and the gonadal sheath cells. TDC-1, but not TBH-1, is expressed in the RIM motor neurons and four UV cells. The co-expression of TDC-1 and TBH-1 suggest that the RIC interneurons and gonadal sheath cells are octopaminergic, whereas the RIM motor neurons and UV cells are tyraminergic. C. elegans foraging behavior consists of forward locomotion interrupted by brief reversals. tdc-1 mutants, unlike tbh-1 mutants, have a marked increase in spontaneous reversals, suggesting that tdc-1 plays a role in the regulation of backward and forward locomotion. Foraging is usually accompanied by oscillatory head movements. In wild-type animals these head movements are suppressed when backward locomotion is induced by anterior touch. tdc-1 but not tbh-1 mutants fail to suppress head movements during backward locomotion. We characterized the neural circuit underlying the modulation of foraging behavior and head movements by laser ablation studies. Ablations of the RIMs but not the RICs increased spontaneous reversals. In addition, animals in which the RIMs are ablated fail to suppress head movements upon anterior touch. The RIMs synapse onto the head muscles and the RMD and SMD motor neurons. The RIMs also synapse onto the AVB forward command neurons and are connected by gap junctions to the AVA backward command neurons. Our ablation experiments indicate that the RIMs link suppression of head movements to a backing response mediated by anterior touch. We hypothesize that tyramine controls foraging and head movements by acting as an inhibitory neurotransmitter on the head muscles and interneurons that coordinate backward and forward locomotion. To identify new factors that function in tyraminergic signaling we screened for mutants that fail to suppress head movements upon anterior touch. We have identified over 50 mutants, which we are characterizing and assigning to complementation groups.

Pirri, Jennifer K et al. (2013) International Worm Meeting "Synaptic engineering: an ionic switch of behavior."

Alkema, Mark J, Rayes, Diego, Pirri, Jennifer K

[International Worm Meeting, 2013]

The unraveling of the human brain connectivity map is considered by many as an essential step in the understanding how the brain controls behavior and how brain malfunction underlies behavioral disorders. Although the behavioral output of neural networks depends on a delicate balance between excitatory and inhibitory synaptic connections, the neural connectivity map does not carry information about the sign of synaptic connections.. Is it possible to reverse the behavioral output of a neural circuit by changing the sign of a synapse? Does the sign of a synapse provide constraints to the development, the specification, and behavioral output of a connectome? Here, we address these questions using the neuronal circuit that mediates the C. elegans escape response. In the escape response a single pair of tyraminergic neurons coordinate the suppression of head movements with backward locomotion through the activation of an inhibitory postsynaptic receptor, the tryamine-gated chloride channel LGC-55. LGC-55 is a member of the Cys-loop ligand gated ion channel (LGIC) family, whose ion selectivity is determined by the M2 domain. Amino acid substitution of the M2 domain LGC-55 allowed us to change the selectivity from Cl- to Na+ and K+. We generated transgenic animals that express the excitatory LGC-55 cation channel under control of its endogenous promoter in an LGC-55 anion mutant background. A fluorescently tagged LGC-55 cation channel localizes opposite to tyramine release sites indistinguishable from the wild-type LGC-55 anion channel. Exogenous tyramine induces neck muscle relaxation and backward locomotion in the wild-type LGC-55 anion animals, but induces neck muscle contraction and forward locomotion in transgenic animals that express the LGC-55 cation. Similarly, touch or optogentically induced release of endogenous tyramine triggers opposite behavioral responses in animals that express the LGC-55 cation vs LGC-55 anion channel. Our data show that changing the nature of a synapse within a neural circuit can reverse its behavioral output and indicate that the C. elegans connectome is established independent of the nature of synaptic activity or behavioral output.

Maguire, Sean et al. (2011) International Worm Meeting "Behavioral adaptation of soil nematodes facilitates escape from predacious fungi."

Clark, Christopher M., Maguire, Sean, Alkema, Mark J., Pirri, Jennifer K.

[International Worm Meeting, 2011]

Animal nervous systems need to respond to challenges in their natural environment. This is epitomized by predator-prey interactions where survival depends on the animal's ability to escape. However, we have few insights into how natural selection has shaped the molecular and neural mechanisms of behavior. We investigated predator-prey interactions between C. elegans and predacious fungi that catch and devour nematodes using constricting rings as trapping devices. Gentle touch to the anterior half of the body of C. elegans induces an escape response in which the animal reverses and suppresses exploratory head movements. We found that the constricting rings of Drechslerella doedycoides catch early larval stages with a diameter similar to the trap opening. There is a delay between the ring entry and ring closure, which allows the animal to withdraw from the trap before getting caught. Tyramine-signaling mutants that fail to suppress head movements in response to touch are caught more efficiently than the wild type in constricting fungal rings. Competition experiments show that the suppression of head movements in response to touch is an ecologically relevant behavior that allows the C. elegans to smoothly retract from a fungal noose and evade capture. Behavioral analysis of other soil nematodes suggests that selective pressures imposed by predacious fungi have shaped the evolution of escape behavior of free-living soil nematodes.

Florman, Jeremy T. et al. (2021) International Worm Meeting "Chemical in vivo activation of C. elegans neurons using a histamine-gated cation channel"

Francis, Michael M., Florman, Jeremy T., Araya Budnik, Antonia, Alkema, Mark J.

[International Worm Meeting, 2021]

The ability to non-invasively activate and inactivate neurons in intact behaving animals provides powerful methods to study the functions of neural circuits. Recently, transgenic expression of a Drosophila melanogaster histamine-gated chloride channel (HisCl1) in C. elegans has enabled hyperpolarization of specific neurons in response to histamine (Pokala et al., 2014). The small molecule histamine is used as a transmitter in many species however C. elegans lack endogenous histamine signaling. We will present data to show that modification of the pore domain of this histamine-gated channel renders it selective to cations rather than anions. We find that cell specific expression of this modified channel, which we named HisCat1, enables histamine induced activation of neurons and can elicit specific behaviors. Pan-neuronal expression of HisCat1 induced uncoordinated seizure like behavior, whereas expression in cholinergic neurons caused hypercontraction and inhibited locomotion in animals exposed to histamine. When we expressed HisCat1 in serotonergic neurons, histamine exposure induced persistent pharyngeal pumping in the absence of food, consistent with the serotonin release. Finally, we demonstrate that HisCat1 can be used to depolarize non-neuronal tissues as expression in body-wall muscle induces calcium influx in response to histamine. Chemical activation using the HisCat1 channel provides a useful complement to existing optogenetic tools. Neuronal activation using HisCat1 appears to be stable over long durations, allowing prolonged activation that is more difficult to achieve with existing optogenetic methods. Furthermore, the HisCat1 channel can be combined with Channelrhodopsin and optical sensors such as GCaMP, so light and histamine can be used to activate and monitor several neurons independently in the same experiment. Therefore, HisCat1 is an effective means to non-invasively activate neurons in C. elegans and increases the existing toolkit of light induced sensors and ion channels. Pokala, N., Liu, Q., Gordus, A., Bargmann, C.I., 2014. Inducible and titratable silencing of Caenorhabditis elegans neurons in vivo with histamine-gated chloride channels. Proc. Natl. Acad. Sci. U. S. A. 111, 2770-5.

David J Katz et al. (2005) International Worm Meeting "Investigating the link between NANOS and chromatin for germ cell maintenance in C. elegans"

William G Kelly, David J Katz

[International Worm Meeting, 2005]

In Drosophila, nanos is required for primordial germ cells (PGCs) to differentiate into a functional germ line. Lack of NANOS function causes defects in germ cell migration, differentiation, and quiescence and ultimately leads to sterility in both males and females. Similarly, in mice a lack of nanos3 results in the complete loss of germ cells in both sexes (Tsuda et al. 2003) and functional loss of two of the three nanos genes in C. elegans, nos1 and nos2, also results in sterility (Subramaniam and Seydoux, 1999). It has therefore been proposed that nanos is a conserved master regulator of the germ cell fate. In the C. elegans early embryo, the chromatin in all cells contain the active chromatin mark, histone H3 methylated on lysine 4 (H3meK4). However upon their birth, the primordial germ cells Z2 and Z3 dramatically lose this mark. This loss of the active H3meK4 mark has been suggested to be part of a chromatin-based transcriptional repression mechanism. Remarkably, absence of the H3meK4 mark has been shown to be dependent upon nanos activity (Schaner et al. 2003). Thus it is possible that nanos may be partially affecting its role in maintaining the undifferentiated germ cell fate through a chromatin-based mechanism. We aim to explore this potential link by further characterizing the role of C. elegans nanos in germ cell fate. To this end, we are taking both forward and reverse genetic approaches as well as conducting ectopic expression studies.

Jun-ichi Aikawa (2001) International C. elegans Meeting "Molecular characterization of heparan sulfate/heparin N-deacetylase/N-sulfotransferase in worms"

Jun-ichi Aikawa

[International C. elegans Meeting, 2001]

Heparan sulfate binds and activates a large variety of growth factors, enzymes and extracellular matrix proteins. These interactions largely depend on the specific arrangement of sulfated moieties and uronic acid epimers within the chains. These oligosaccharide sequences are generated in a step-wise manner, initiated by the formation of a linkage tetrasaccharide which is then extended by copolymerization of alternating a1,4GlcNAc and b1,4GlcA residues. As the chains polymerize, they undergo a series of sulfation and epimerization reactions. The first set of modifications involves the removal of acetyl units from subsets of GlcNAc residues, and the addition of sulfate groups to the resulting free amino groups. These reactions are catalyzed by a family of enzymes designated as GlcNAc N-deacetylase/N-sulfotransferases (NDST), since they simultaneously. Four members of the family have been identified in vertebrates, with single orthologs present in Drosophila and C. elegans. We have revealed tissue-specific expression pattern and unique enzymatic properties of these four isozymes1,2). In fly, loss of NDST (sulfateless) results in unsulfated chains and defective signaling by multiple growth factors and morphogens. I reconstituted cDNA for worm NDST from EST clones and 5' RACE products. Enzymatic activities will be discussed. 1) Aikawa, J. & Esko, J. D J. Biol. Chem. 274, 2690-2695 (1999) 2) Aikawa, J., Grobe, K., Tsujimoto, M. & Esko, J. D J. Biol. Chem. 276, 5876-5882 (2001)

Snyder, Matthew P. et al. (2013) International Worm Meeting "Turnover of the H3K9me2 Mark During Late Spermatogenesis."

Snyder, Matthew P., Xu, Xia, Maine, Eleanor

[International Worm Meeting, 2013]

Meiotic silencing is a conserved phenomenon targeting unpaired chromosomes and chromosomal regions during prophase of meiosis I. Meiotic silencing in animals typically occurs at the chromatin level and involves accumulation of histone modifications thought to promote a closed chromatin configuration. This chromatin structure may contribute to transcriptional repression and meiotic chromosomal events such as chromosome disjunction (Bean et al 2004, Jaramillo-Lambert and Engebrecht 2010). During meiosis in C. elegans, non-synapsed chromosomes are enriched for H3K9me2 relative to synapsed chromosomes (Kelly et al. 2002; Bean et al. 2004). Such non-synapsed chromosomes include the male X, homologous chromosomes that fail to synapse due to mutation, and chromosomal translocations/duplications. The pattern of H3K9me2 accumulation during meiosis depends on activity of the small RNA machinery, which may have a role in targeting the mark to unpaired chromosomes and ensuring that the mark does not persist abnormally (Maine et al. 2005; She et al. 2009). Taking a combined biochemical/genetic approach, we are identifying additional factors important for regulating H3K9me2 distribution during meiosis. Through this approach, we are identifying genes that appear to be important for turnover of the H3K9me2 mark during late spermatogenesis.

Husson, Steven J. et al. (2011) International Worm Meeting "C. elegans mutants defective in neuropeptide amidation enzymes are abnormal in egg-laying behavior."

Landuyt, Bart, Schoofs, Liliane, Gottschalk, Alexander, Horvitz, H.Robert, Temmerman, Liesbet, Husson, Steven J., Ringstad, Niels, Meelkop, Ellen

[International Worm Meeting, 2011]

Egg laying has mainly been studied at the behavioral, neuronal and neurochemical levels, but little is known about the biochemical control of the relevant neuropeptidergic signaling systems. Biosynthesis of endogenous peptides requires processing enzymes, such as proprotein convertase 2, which is encoded by egl-3 (1, 2), and a carboxypeptidase encoded by egl-21 (3, 4). Mutants defective in these genes have egg-laying defects, consistent with the finding that FMRFamide-like peptides (FLPs) have been linked to egg laying behavior. C. elegans enzymes that carry out the last step in the production of biologically active peptides, the carboxy-terminal amidation reaction, have not been characterized. This multistep reaction involves hydroxylation of the glycine a-carbon by a peptidyl-a-hydroxylating monooxygenase (PHM), followed by a cleavage reaction performed by peptidyl a-hydroxyglycine a-amidating lyase (PAL) to generate a glyoxylate molecule and the a-amidated peptide. In vertebrates, both enzymatic activities responsible for the carboxyterminal amidation reaction are contained in one bifunctional enzyme, peptidylglycine a-amidating monooxygenase (PAM). By contrast, invertebrates generally express two separate enzymes encoded by two different genes. Here we report the identification and characterization of C. elegans amidating enzymes using bioinformatics to identify candidate genes and mass spectrometry to compare the neuropeptides in wild-type and newly generated mutants. Mutants lacking a functional PHM displayed an altered neuropeptide profile, showed impaired egg laying behavior and had a decreased brood size. Interestingly, PHM mutants still displayed fully processed amidated neuropeptides, probably as a result of the presence of a bifunctional PAM, the main amidating enzyme in vertebrates. Our data indicate the existence of a robust complementation system for the amidation reaction of neuropeptides in nematodes and suggest the involvement of amidated neuropeptides in egg laying. (1) S. J. Husson et al., J. Neurochem. 98, 1999 (2006); (2) J. Kass et al., J. Neurosci. 21, 9265 (2001); (3) S. J. Husson et al., J. Neurochem. 102, 246 (2007); (4) T. C. Jacob, J. M. Kaplan, J. Neurosci. 23, 2122 (2003).

Crittenden SL et al. (1997) International C. elegans Meeting "TEMPERATURE-SENSITIVE REPRESSION OF TRANSGENES CARRYING A fem-3(gf) MUTANT 3'UTR"

Crittenden SL, Kimble JE

[International C. elegans Meeting, 1997]

We are developing a method to control gene expression in a temperature dependent manner. Our strategy is to regulate reporter expression with a well-defined promoter at the 5' end and a mutant fem-3 3'UTR at the 3' end. The fem-3(gf) mutations map to the fem-3 3'UTR and release fem-3 from post-transcriptional repression at 25 C, but not at 15 C (1,2). We have tested expression of a lag-2 promoter::GFP construct that carries either a strong or a weak fem-3(gf) 3'UTR. The lag-2 promoter drives expression in the distal tip cells of adults (3-5). We find that transgenes express GFP at much lower levels at 15 C than at 25 C when carrying a fem-3(gf) 3'UTR. We are currently trying to optimize repression at 15 C using the lag-2 promoter and GFP as a reporter. In addition, we are testing whether temperature sensitive repression can be achieved in other tissues, and we are starting to explore the biological activity of various proteins expressed in the distal tip cell. 1. Ahringer, J., and Kimble, J. (1991). Nature 349, 346-348. 2. Barton, M. K., Schedl, T. B., and Kimble, J. (1987). Genetics 115, 107-119. 3. Fitzgerald, K., and Greenwald, I. (1995). Development 121, 4275-4282. 4. Gao, D. and J. Kimble, midwest worm meeting 1996. 5. Henderson, S. T., Gao, D., Lambie, E. J., and Kimble, J. (1994). Development 120, 2913-2924.

Fujita, Kosuke et al. (2011) International Worm Meeting "Dopamine signaling for regulating experience-dependent odor avoidance."

Fujita, Kosuke, Kimura, Kotaro

[International Worm Meeting, 2011]

Dopamine signaling plays significant roles in regulating behavior and in learning. The underlying in vivo molecular mechanisms, however, are complex and still unclear. We have reported that the D2-like dopamine receptor DOP-3 is required in the RIC interneurons to regulate the enhancement to a repulsive odor 2-nonanone in worms (Kimura, Fujita and Katsura, J. Neurosci., 2010). Avoidance behavior of worms to 2-nonanone is significantly enhanced, rather than reduced, after 1 hr-preexposure to the odor. Unlike previously identified dopamine-regulated behavioral plasticities in worms, which require the presence of food for dopamine action (Sawin et al., Neuron, 2000; Hills et al., J. Neurosci., 2004; Sanyal., EMBO J., 2004; Kindt et al., Neuron, 2007), the enhancement of 2-nonanone avoidance is observed even after food absence for 2 hr, suggesting a novel role for dopamine signaling. In addition to the RIC-specific rescue of the dop-3 phenotype, we recently found that RIC-specific RNAi of dop-3 suppresses the enhanced avoidance behavior, indicating that DOP-3 activity in RIC is necessary and sufficient to regulate the behavioral plasticity (K. F. and K. K, unpublished). How does dopamine signaling in RIC regulate the enhancement of 2-nonanone avoidance? The RIC neurons are known as octopaminergic, and octopamine release is suppressed by dopamine signaling via DOP-3 to regulate CREB expression in the SIA neurons (Alkema et al., Neuron, 2005; Suo et al., EMBO J., 2009). To test the involvement of octopamine signaling in the enhancement of 2-nonanone avoidance, we investigated if the dop-3 phenotype is suppressed by null mutations in tbh-1, a tyramine beta-hydroxylase required for octopamine synthesis in RIC. The double mutants tbh-1(n3247)dop-3(tm1356) and tbh-1(ok1196)dop-3(tm1356) showed partial suppression of the enhancement-defective phenotype, suggesting that the antagonistic octopamine signaling is a part of, but not all of, the output for the dopamine signaling in RIC to regulate the enhancement. In addition to identifying the octopamine receptor(s) required for signaling, we are currently attempting to identify the other neuronal outputs from RIC by RIC-specific RNAi.