Kerr, Rex et al. (2019) International Worm Meeting "Bridging the gap from raw data to result in large-scale aging experiments."

Kenyon, Cynthia, Kerr, Rex, Kimmel, Jacob, Xue, Eddie

[International Worm Meeting, 2019]

Animals exhibit declining behavioral capacity as they age. These declines typically include a reduction of speed and strength of movement, slowed reactions, and loss of coordination. However, the genes involved in preserving youthful behavior or setting the pace and nature of the decline have not been extensively studied, in large part due to the difficulty of conducting and analyzing experiments on a sufficient scale. We have created an automated system, the C. elegans Observatory, to simultaneously characterize hundreds of strains of worms as they age by computing a variety of behavioral metrics. However, analyzing the resulting data becomes an intractable burden for a typical experimentalist. We therefore have constructed a series of automated analyses, ranging from low-level through to near-publication-quality graphs for key behaviors, which run on the fly as data is generated. This enables the same kind of rapid understanding and iterative experimentation in a big-data style experiment as is typical for a benchtop experiment. Examples of the analyses, using data from ongoing experiments on wild-type and long-lived mutants, will be used to illustrate the process.

L'Etoile ND et al. (1997) International C. elegans Meeting "ODR-1 ENCODES A RECEPTOR GUANYLYL CYCLASE"

Bargmann CI, L'Etoile ND

[International C. elegans Meeting, 1997]

odr-1 is required by C. elegans to detect specific odorants in its environment. odr-1 mutants fail to chemotax towards odorants, such as benzaldehyde, butanone and isoamyl alcohol, which are known to be recognized by chemosensory neuron AWC. However, odr-1 mutants respond normally to volatile chemoattractants sensed by AWA, the other chemosensory neuron that mediates attraction to volatile compounds. We rescued the chemotaxis defect of odr-1 mutants by germline transformation with an 8.5 kb genomic fragment containing a predicted receptor guanylyl cyclase gene (RGC). Sequence analysis of the odr-1(ky29) allele revealed a nonsense mutation within the first half of the RGC gene, making ky29 a good null mutation candidate. An ODR-1::GFP fusion is expressed in AWC as well as chemosensory neurons AWB, ASI, ASK and ASJ. This correlates with other odr-1 phenotypes which include loss of AWB mediated avoidance of octanol (B. Kimmel, unpublished data) and a synthetic dauer constitutive phenotype of an ASI defect (Katsura et al., worm meeting, 1995). We are investigating whether ODR-1 functions in the chemosensory signal transduction pathway, perhaps by opening the TAX-2 /TAX-4 cGMP-gated cation channel (which is also required for AWC function) via production of cGMP. The biochemistry of RGC action has been studied in detail. We hope to use information about the biochemistry of RGCs to determine which aspects of RGC function are required in C. elegans sensory signal transduction.

Redo Riveiro, Alba et al. (2013) International Worm Meeting "egl-46 is a novel BAG cell fate modulator."

Pocock, Roger, Redo Riveiro, Alba

[International Worm Meeting, 2013]

Genetic studies in C. elegans attempt to identify molecular components required for neural circuits to function correctly. This study focuses on BAG neurons, which have the major role in oxygen (O2) and carbon dioxide (CO2) sensing. Development of BAG neurons is controlled by the conserved transcription factors ETS-5 (Brandt, Aziz-Zaman et al. 2012) and EGL-13 (Petersen et al. 2013-in press). However the effect of ets-5 and egl-13 loss of function mutants does not affect all BAG terminal fate markers. Therefore, other genes must act in parallel to these factors to specify BAG fate. In a classical forward genetic screen, we identified egl-46 as a BAG fate modulator. EGL-46 is a zinc finger transcription factor that regulates terminal cell divisions in the Q lineage where egl-46 mutants undergo extra rounds of terminal Q cell divisions (Wu, Duggan et al. 2001). The egl-46 mammalian ortholog Insm1 has a similar function in determining cell fates. Insm1 has been implicated in the development of pancreas (Farkas, Haffner et al. 2008), cortex and hindbrain (Jacob, Storm et al. 2009). Insm1 mutant mice present a thicker layer of proliferative progenitors and a thinner neuro-basal layer in the olfactory ephitelium (Rosenbaum, Duggan et al. 2011). Here we show that egl-46 mutants have defects in the expression of specific terminal markers in the BAG neurons. We have rescued these defects by transgenically expressing a fosmid containing the egl-46 locus. Currently, we are performing genetic interaction studies with BAG-fate regulators like ets-5 and egl-13.

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).

Hung, Wesley et al. (2011) International Worm Meeting "An F-box protein FSN-1 Regulates Retrograde Insulin Signalling."

Hwang, Christine, Chitturi, Jyothsna, Li, Hang, Wang, Ying, Hung, Wesley, Zhen, Mei, Liao, Edward

[International Worm Meeting, 2011]

The formation of a functional synapse requires the co-ordinated growth of both pre-synaptic and post-synaptic termini. Communication between the pre- and post-synaptic termini involves many proteins and factors. The pre-synaptic F-box protein FSN-1 is an evolutionarily conserved protein that plays an important role in axon growth and synapse development. FSN-1 is a part of an SCF-like E3 ubiquitin ligase complex that comprises of the RING finger domain protein RPM-1, Cullin and Skp (1). FSN-1/RPM-1 complex negatively regulates a MAPK kinase cascade comprised of DLK-1/MKK-4/PMK-3 during synapse formation (2). We report here that FSN-1 also regulates synapse development, in part, through the regulation of a retrograde insulin/IGF-signalling pathway in the post-synaptic terminal. Mutation in fsn-1 causes over-development of some synapses along the dorsal nerve cord while other areas of the dorsal cord have little or no synapses. In fsn-1 mutants there is an increased activity of the insulin/IGF signalling pathway in the post-synaptic muscle cells. Accordingly, loss-of-function mutations in the insulin/IGF pathway components suppressed the synaptic morphology defects of fsn-1 mutants. We identified several neuronal insulin-like ligands that regulate the postsynaptic insulin/IGF signal during synapse development; all require a proprotein convertase, EGL-3 (3, 4), for maturation. We further demonstrate that, in vivo, EGL-3::GFP's level is increased in fsn-1 mutants. in vitro, FSN-1 interacts with, and ubiquitinates EGL-3. We propose that FSN-1 modulates the activity of the post-synaptic insulin/IGF pathway via its regulation of EGL-3. 1. Liao, E.H., W. Hung, et al. (2004). An SCF-like ubiquitin ligase complex that controls presynaptic differentiation. Nature 430: 345-350. 2. Nakata K, B. Abrams , et al. (2005). Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development. Cell 120:407- 420. 3. Kass, J., T.C. Jacob, et al. (2001).The EGL-3 proprotein convertase regulates mechanosensory responses of Caenorhabditis elegans. J Neurosci 21: 9265-9272. 4. Husson, S. J., T. Janssen, et al. (2007). Impaired processing of FLP and NLP peptides in carboxypeptidase E (EGL-21)-deficient Caenorhabditis elegans as analyzed by mass spectrometry. J Neurochem 102: 246-60.

Patricia R Goodwin et al. (2007) International Worm Meeting "CDK-5 regulates synaptic transmission at the neuromuscular junction."

Gian Garriga, Peter Juo, Tom Harbaugh, Patricia R Goodwin

[International Worm Meeting, 2007]

CDK-5 is a cyclin-dependent kinase that acts primarily in the nervous system, where it is involved in multiple aspects of neuronal development, contributes to neurodegeneration, and has recently emerged as a regulator of synaptic plasticity (1,2). We examined the role of CDK-5 in synaptic transmission at the C. elegans neuromuscular junction (NMJ) by testing cdk-5 mutants for changes in paralysis induced by the acetylcholine (ACh) esterase inhibitor aldicarb. We show here that two independent loss-of-function alleles of cdk-5, gm336 and ok626, are resistant to aldicarb-induced paralysis. Both alleles result in deletions that remove the start codon and are predicted to be molecular nulls. Expression of wild type cdk-5 cDNA under the cholinergic motorneuron promoter (Punc-17) rescues the decrease in synaptic transmission observed in cdk-5 null mutants. Furthermore, overexpression of cdk-5 under the unc-17 promoter in wild type animals results in increased synaptic transmission. CDK-5 activity is dependent on activation by its binding partner, p35 (1). Consistent with this, we find that p35(gm335) loss-of-function mutants are resistant to aldicarb-induced paralysis. In order to identify how CDK-5 is regulating synaptic transmission, we are currently analyzing the effect of cdk-5 mutations on a panel of fluorescent presynaptic markers. We are also investigating the possibility that CDK-5 may indirectly influence ACh release by regulating neuropeptide secretion. Neuropeptides can modulate ACh release and mutations in neuropeptide processing enzymes, such as the proprotein convertase egl-3 PC2, are resistant to aldicarb-induced paralysis (3). Preliminary evidence shows that cdk-5(gm336); egl-3(nr2090) double mutants have rates of aldicarb-induced paralysis that are comparable to either single mutant alone, suggesting that CDK-5 and PC2 act in the same pathway to increase ACh release. We are currently testing other genes involved in neuropeptide processing for genetic interactions with cdk-5 and directly imaging fluorescently tagged neuropeptides (4) in cdk-5 mutants. Results from these various experiments will be presented. (1) Dhavan, R. and Tsai, L.H. (2001). Nat. Rev. Mol. Cell. Biol. 2: 749-759. (2)Cheung, Z.H. et. al. (2006). Neuron 50: 13-18. (3) Jacob, T.C. and Kaplan, J.M. (2003). J. Neurosci. 23:2122-2130. (4) Sieberth, D.S. et. al. (2006). Nat Neurosci. 10: 49-57.

Makoto Tsunozaki et al. (2004) West Coast Worm Meeting "An odr-11 mutation reverses AWC-mediated butanone responses from attraction to avoidance"

Cori Bargmann, Makoto Tsunozaki

[West Coast Worm Meeting, 2004]

When wild-type C. elegans is exposed to a particular odor, the identity of the cell that senses the odor specifies the behavioral response. C. elegans has three pairs of olfactory neurons: AWA, AWB and AWC. AWA and AWC mediate attraction, and AWB mediates an avoidance response. It has been shown that ectopic expression, in AWB, of an odorant receptor for a normally attractive odor can reprogram the animal's response to the ligand from attraction to avoidance (Troemel et al., 1997). Here we describe a mutant, odr-11(ky713) , in which AWC mediates an avoidance response to a normally attractive odor, butanone. odr-11 animals are defective in responses to AWC-sensed odors but have normal responses to AWA- and AWB-sensed odors. Mutant animals avoid butanone and show reduced chemotaxis to benzaldehyde, isoamylalcohol, and 2,3-pentanedione. Manipulating the number of AWC cells that sense butanone quantitatively alters butanone avoidance. In wild-type animals, one AWC neuron senses butanone. ceh-36 mutants have no AWC neurons, and nsy-1 mutants have two butanone-sensing AWC neurons. Butanone avoidance is absent in odr-11;ceh-36 mutants, and is enhanced in odr-11;nsy-1 mutants compared to odr-11 single mutants. These results suggest that abnormal AWC activity in odr-11 mutants causes butanone avoidance. Currently, we are characterizing the genetic interaction of odr-11 with other mutations known to affect chemotaxis in AWC. Preliminary data suggest that the guanylyl cyclase ODR-1 is required for butanone avoidance. To further understand the nature of the defects in chemotaxis mutants, we are carrying out quantitative analysis of behavioral responses to odors using an odor flow chamber. The flow chamber allows us to create a temporal change in odor concentration while the animals are being filmed. Wild-type animals respond to a step increase in butanone concentration by suppressing reversals, while they respond to a step decrease by increasing the frequency of reversals. These observations are consistent with the biased random-walk model for chemotaxis (Pierce-Shimomura et al., 1999). Animals respond to a short impulsive stimulus of butanone by an initial suppression of reversals followed by an increase in reversals above baseline. A preliminary analysis of the temporal profile of the response suggests that animals have a signal integration time on the order of tens of seconds. We are examining various chemotaxis mutants, including odr-11 , to further elucidate the role of each gene in the behavioral response to odors. References: Troemel, E.R., Kimmel, B. E. and Bargmann, C. I. (1997) Cell 91 :161-169. Pierce-Shimomura, J. T., Morse, T. M., Lockery, S. R. (1999) J. Neurosci. 19 :9557-9569.

Goodwin, Patricia R. et al. (2009) International Worm Meeting "CDK-5 regulates polarized trafficking of dense core vesicles."

Juo, Peter, Goodwin, Patricia R.

[International Worm Meeting, 2009]

Neuropeptides are neuromodulators that regulate classical neurotransmitter activity in mammals and invertebrates. Neuropeptides are released from dense core vesicles (DCVs), which can undergo polarized trafficking to the axon and release at synaptic sites. In C. elegans, neuropeptide release facilitates cholinergic synaptic transmission at the neuromuscular junction (1). DCVs can be visualized in vivo using fluorescently-tagged neuropeptides (such as INS-22::Venus)(2,3). In polarized DA motorneurons, DCVs are primarily localized to the axon in the dorsal nerve cord by the kinesin motor UNC-104/Kif1A (1-4). In this study, we show that cyclin dependent kinase-5 (CDK-5) functions in cholinergic motorneurons to regulate the polarized trafficking of DCVs to axons. In mammals, CDK-5 and its cyclin-like activator, p35, regulate multiple cellular functions including axon outgrowth and synaptic transmission, and dysregulation of CDK-5 contributes to several neurodegenerative disorders. Here we show that in cdk-5(gm336) or p35(tm648) loss-of-function mutants, the abundance of DCVs in the axons of DA motorneurons decreases with a coincident increase in the abundance of DCVs in the dendrite. This increase in dendritic DCVs can be rescued by expressing wild type cdk-5 cDNA in cholinergic neurons. The effect of CDK-5 on polarized DCV trafficking is relatively specific since the presynaptic localization of synaptic vesicle marker SNB-1::GFP and active zone marker SYD-2::GFP is not altered in cdk-5 mutants. To investigate why DCVs accumulate in the dendrites of cdk-5 null mutants, we performed time-lapse imaging of mobile INS-22::Venus puncta in motorneuron dendrites. DCVs are highly mobile and move in a saltatory fashion in both anterograde and retrograde directions (4). Our data indicate that in the DA motorneuron dendrite of cdk-5 mutants, there is an increase in the total number of DCVs moving in the anterograde direction and an increase in total number of stationary DCVs, compared to wild type controls. There is no change in the number of DCVs moving in the retrograde direction. The ectopic DCVs observed in the dendrites of cdk-5 mutants do not appear to be transported there by their normal axonal motor, UNC-104/Kif1A, because deletion of cdk-5 in an unc-104 null background still results in an increase the number of DCVs in the dendrite. We hypothesize that CDK-5 regulates polarized DCV trafficking by inhibiting the inappropriate loading of DCVs onto an unidentified dendrite-directed motor. References: (1)Jacob & Kaplan. J Neurosci 2003 (2)Sieberth et al. Nature 2005 (3)Sieberth et al. Nat Neurosci 2006 (4)Zahn et al. Traffic 2004 (5)Dhavan and Tsai. Nat Reviews 2001.