Feng, Weidong et al. (2021) International Worm Meeting "A noncanonical role for Hox in the C. elegans ventral nerve cord"

Feng, Weidong, Kratsios, Paschalis

[International Worm Meeting, 2021]

Hox proteins constitute a phylogenetically conserved family of transcription factors that play essential roles during early animal development, such as control of regional identity and cell survival. However, our understanding of Hox gene function during post-embryonic stages remains rudimentary. To bridge this gap, we focused on the C. elegans nerve cord, which is populated by eight different classes of motor neurons (MNs) - the cholinergic DA, DB, VA, VB, AS, VC and the GABAergic DD and VD neurons. By generating an endogenous reporter allele for the mid-body Hox gene lin-39 (Scr/Dfd/Hox4-5), we found that it is continuously expressed - from developmental to adult stages - in MNs of all these eight classes. Using null and auxin-inducible alleles, we observed that lin-39 is required not only to establish, but also maintain in the adult the expression of effector molecules that define MN final (terminal) identity, such as neurotransmitter biosynthesis proteins, ion channels and adhesion molecules. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) for LIN-39 together with mutational analysis of its cognate binding site strongly suggest that it acts directly to activate expression of its target genes. LIN-39 does not act alone. Our genetic and biochemical studies indicate that it collaborates with two terminal selector-type transcription factors: UNC-3/Ebf in cholinergic MN classes (DA, DB, VA, VB, AS) and UNC-30/Pitx in GABAergic MNs (DD, VD). We also found that LIN-39 partners with another Hox gene (mab-5/Antp/Hox6-8) to consolidate the identity of both cholinergic and GABAergic MNs. Our genetic and biochemical analyses suggest that the expression of lin-39 and mab-5 in adult nerve cord MNs is maintained via direct transcriptional autoregulation. Intriguingly, lin-39 and mab-5 can jointly control the expression levels of unc-3 in cholinergic MNs, thereby generating a coherent feed forward loop-type of mechanism (i.e., Hox and unc-3 activate the same effector molecules, but Hox proteins also activate unc-3) for the establishment and maintenance of motor neuron identity. Based on recent profiling studies showing Hox gene expression in the adult fly and mouse nervous systems, the noncanonical Hox gene function described here may be phylogenetically conserved.

Feng, Weidong et al. (2021) International Worm Meeting "The C. elegans Hox gene ceh-13/labial/Hox1 controls motor neuron terminal identity"

Kratsios, Paschalis, Feng, Weidong

[International Worm Meeting, 2021]

The phylogenetically conserved Hox gene family is known for its early roles in embryonic patterning along the anteroposterior axis. However, whether Hox genes exert later roles during development and post-embryonic life, remains unclear. In the context of the nervous system, Hox genes control fundamental processes occurring during early development, such as neuronal specification and axon guidance. On the other hand, we know much less about the function of Hox genes in the last steps of neuronal development, during which neurons obtain their terminal identity features, such as expression of neurotransmitters, receptors and ion channels. Among the six C. elegans Hox genes, the function of ceh-13/labial/Hox1 remains poorly understood, in part due to the early embryonic/larval lethality observed in ceh-13 null mutant animals. Here, we generated a conditional allele based on the auxin-inducible protein degradation system, enabling post-embryonic CEH-13 depletion. This allele also serves as an endogenous ceh-13 reporter, enabling us to establish, with single cell resolution, the expression profile of ceh-13 in post-mitotic larval motor neurons of the ventral nerve cord. Using ceh-13 null mutants, we identified three terminal identity genes (acr-2/CHRNA1, unc-53/NAV1, unc-129/GDF10) as CEH-13 targets, suggesting that it controls the establishment of motor neuron terminal identity. We also observed lifelong expression of ceh-13 in motor neurons, suggesting that it is required to maintain terminal identity features in these cells. Lastly, we found that expression of a transgenic ceh-13 reporter is significantly reduced in nerve cord motor neurons upon either genetic removal of ceh-13 during early development or inducible CEH-13 depletion at post-embryonic stages, raising the possibility of transcriptional autoregulation as a mechanism for lifelong ceh-13 expression. These findings advance our current understanding of ceh-13/labial/Hox1 function in C. elegans and further suggest that Hox proteins control both early and late steps of neuronal development.

Rauthan, M. et al. (2017) International Worm Meeting "MicroRNA regulation of nicotine-dependent behavior in C. elegans."

Rauthan, M., Ronan, E.A., Gong, J., Xu, X.Z.S., Liu, J., Wescott, S.

[International Worm Meeting, 2017]

Tobacco smoking is the leading cause of preventable death in developed nations. Nicotine is the principle addictive substance in cigarettes. Chronic exposure to nicotine up-regulates nAChRs and is thought to play a critical role in the primary steps of nicotine dependence, but the underlying mechanisms are not well understood. We have previously developed a C. elegans model of nicotine dependent behavior, and shown that the nAChR gene acr-15 is required for acute response to nicotine (Feng et al., 2006). Here we identify a key role for microRNA in regulating nicotine-dependent behavior by modulating nAChR expression in C. elegans. Specifically, we show that chronic nicotine treatment down-regulates microRNA machinery, leading to up-regulation of another nAChR gene that is specifically required for nicotine withdrawal behavior. This effect is mediated by a microRNA that recognizes the 3'UTR of nAChR transcripts. These observations uncover an interesting phenomenon that different nAChRs mediate distinct aspects of nicotine dependence in C. elegans. Our results reveal a functional link between nicotine, microRNA, nAChRs, and nicotine-dependent behavior. Reference(s): 1. Feng, Z., Li, W., Ward, A., Piggott, B.J., Larkspur, E., Sternberg, P.W., and Xu, X.Z.S. (2006). A C. elegans model of nicotine-dependent behavior: regulation by TRP family channels. Cell 127, 621-633.

Christopher J Cronin et al. (2005) International Worm Meeting "The Combined UC San Diego/Caltech Quantitative Behavior Analysis System"

William R Schafer, Zhaoyang Feng, Kuang-man Huang, Paul W Sternberg, Christopher J Cronin

[International Worm Meeting, 2005]

California Institute of Technology and University of California, San Diego have each developed practical, automated movement analysis systems to quantify aspects of worm behavior and morphology (see An imaging system for standardized quantitative analysis of C. elegans behavior, Zhaoyang Feng, et al, BMC Bioinformatics 2004, 5:115, An automated system for measuring parameters of nematode sinusoidal movement, Christopher J Cronin, et al, BMC Genetics 2005, 6:5, and previous meeting abstracts). We have consolidated our development efforts and merged our independent system designs to create a single, unified behavior analysis system. The combined system provides researchers with all of the quantification tools and database functionality of the individual UCSD and Caltech systems, but also establishes a common, open software and hardware foundation for continued development by Caltech, UCSD and the community. Current efforts include expanding system capabilities for the automated analysis of mating behavior with the challenge of tracking multiple moving individuals, both in contact with and separated from each other.

Rauthan, Manish et al. (2009) International Worm Meeting "Role of neurotransmitters and neuropeptides in C. elegans nicotine dependent behavior."

Feng, Zhaoyang, Rauthan, Manish, Xu, X. Z. Shawn

[International Worm Meeting, 2009]

Nicotine is the main addictive drug in tobacco. In mammals, nicotine exerts its effects by binding to nicotinic acetylcholine receptors (nAChRs), which are ligand-gated ion channels and allow both mono- and divalent cations to pass through them. Nicotine acts by increasing the dopamine levels in mesolimbic dopamine system, thereby modulating the brain reward centre. Besides dopamine, other neurotrasmitters such as norepinephrine, serotonin and glutamate are involved in different aspects of nicotine dependence. Previous studies in our laboratory established C. elegans as a genetic model for the study of nicotine dependence (Feng, Z. et al. Cell 2006;127(3):621-33). Nicotine elicits various behavior responses in C. elegans such as acute response, adaptation, withdrawal and sensitization. We have found that neuropeptides are involved in nicotine-dependent behavior in worms. We have also found that various neurotransmitters such as dopamine, octopamine and tyramine are involved in modulating nicotine-dependent behavior in worms. In addition, we are identifying novel genes involved in regulating nicotine responses.

Denis V Touroutine et al. (2007) International Worm Meeting "Genetic screens for mutants with altered ACR-16 nicotinic receptor localization and function."

Janet E Richmond, Joshua Jonson, Denis V Touroutine

[International Worm Meeting, 2007]

Individual C. elegans body wall muscles express two pharmacologically distinct nicotinic receptors: a levamisole-sensitive and a levamisole-insensitive receptor class. We have identified ACR-16 as an essential subunit (Touroutine et.al. JBC) of the levamisole-insensitive receptor. This receptor contributes to synaptic transmission at the C. elegans neuromuscular junction (NMJ) and also functions in neurons to mediate nicotine-dependent behavior (Feng et al., Cell). Several components required to traffic or localize the levamisole-sensitive receptor have been identified. However, molecules required to target the ACR-16 receptor to post-synaptic sites remain to be identified, in part due to the lack of a screenable phenotype in acr-16 mutants. We developed two strategies to identify those molecules. 1. We conducted a genetic screen to identify mutants with altered ACR-16::GFP expression. 2. We generated a strain expressing an ACR-16 gain-of-function receptor which desensitizes slowly and causes lethality when expressed in worm muscles. However, worms grown on plates with RNAi against acr-16 survive and are healthy. This allows as to mutagenise acr-16 RNAi-treated worms and screen for suppressors of the lethal phenotype on OP50 plates. Utilizing the first strategy and after screening 2400 haploid genomes, we identified several candidates in which ACR-16::GFP expression is altered. Among these, we found a new allele of ric-3, a gene that is required for both levamisome-receptor and ACR-16 receptor function (Halevi et. al). Another mutant identified in this screen exhibits a dramatic reduction (only 5% of the signal remains) in the ACR-16::GFP puncta that localize adjacent to UNC-17-positive puncta in the ventral nerve cord. Double mutants of unc-63 and this mutant exhibit decreased thrashing rates (77% less) when compared to unc-63 mutants alone, suggesting they have reduced functional ACR-16 receptors at the NMJ. Mutants isolated from both screens will be mapped and characterized. Touroutine et. al JBC 2005 Jul 22;280(29):27013-21. Feng Z et. al. Cell 127:621-633. Halevi et.al EMBO J, 2002 Mar 1;21(5):1012-20.

Zhaoyang Feng et al. (2003) International Worm Meeting "C. elegans, a genetically tractable model for the study of nicotinic signaling"

Anthony Kempf, William Schafer, Zhaoyang Feng, Marika Orlov

[International Worm Meeting, 2003]

Nicotine abuse is the most critical but preventable public health problem. Although nicotinic acetylcholine receptor (nAChR) is identified as the main target of nicotinic signaling, the molecular and neuronal circuits of nicotinic signaling remain unknown. Using invertebrate models such as c. elegans or Drosophila metanogaster as animal models to study drug abuse has recently attracted the attention of researchers because of their simple nervous system, and the available powerful genetic tools in these models [Schafer WR, J. Neurosciences, 2002, Wolf FW and Heberlein U, Cur. Opp. in Neuroscience, 2003]. Utilizing a quantitative c. elegans behavior analysis system (Feng Z. et al, abstract in this meeting), a c. elegans locomotory acclimation behavior is identified and defined. Using this locomotory acclimation behavior as a behavioral assay, the c. elegans animals were found to response acutely to nicotine at a wild concentration range including concentrations close to physiological concentration in human blood after a cigarette consummation. This locomotory acclimation behavior is also found can be used in the study of nicotine adaptation, withdrawal, and possibly sensitization. The distinct roles of several nicotinic receptor subunits (unc-29, unc-38, and unc-63), Go and Gq signaling circuits, and several neuronal transmitters (dopamine, serotonin, glutamate) in the locomotory acclimation behavior, and their functions in nicotinic signaling are discussed.

Ward, Alex et al. (2009) International Worm Meeting "Eyeless but not blind: Phototransduction in C. elegans."

Liu, Jie, Kang, Lijun, Decaluwe, Brandon, Gao, Jingwei, Xie, Zhixiong, Ma, Di, Ward, Alex, Yu, Yong, Inada, Hitoshi, Mori, Ikue, Xu, X.Z. Shawn, Nishio, Nana

[International Worm Meeting, 2009]

It has long been assumed that the nematode C. elegans lacks the sense of light, mainly because it lives in the soil and does not have eyes. However, we have recently reported the surprising observation that C. elegans in fact possesses a simple visual system and engages in phototaxis behavior that is mediated by photoreceptor cells and light-sensitive channels [1]. Here we elucidate the phototransduction cascade in C. elegans photoreceptor cells through a combination of electrophysiological and behavioral analysis. As is the case with vertebrate photoreceptor cell rods and cones, C. elegans phototransduction is also mediated by G signaling and cGMP-sensitive CNG channels. Interestingly, instead of signaling through phosphodiesterases (PDEs), light-activated G proteins appear to be coupled to guanylate cyclases that produce cGMP, thereby resulting in opening of CNG channels. Our studies identify a new sensory modality in C. elegans and suggest that animals living in dark environments (e.g. soil and caves) may not be presumed to be blind. Our data also reveal a surprising conservation in phototransduction between vertebrates and C. elegans, indicating that C. elegans represents a powerful genetic model for the study of phototransduction. [1] Ward, A.*, Liu, J.*, Feng, Z., and Xu, X.Z.S. (2008) Light-sensitive neurons and channels mediate phototaxis in C. elegans. Nature Neuroscience 11, 916-22 *co-first authors.

Kuang-man Huang et al. (2005) International Worm Meeting "Cluster Analysis of Worm Behavior"

Kuang-man Huang, Kathleen Quast, Kathy Andrews, William R Schafer, Pamela Cosman

[International Worm Meeting, 2005]

Over 100 genes have been described in which mutations lead to abnormal or uncoordinated movement (Genetics; Brenner et al. 1974). Uncoordinated (Unc) mutants usually are classified into a number of different descriptive categories, including kinker, coiler, slow, sluggish, and loopy (Genetics; Hodgkin 1983). We have been exploring the use of machine vision to generate more precise quantitative definitions of uncoordinated phenotypes that can be used for bioinformatic studies. Building on previously developed automated tracking systems (Baek et al, 2002; Feng et al., 2004, Cronin et al., 2005), we have developed image processing algorithms to analyze the body postures of worms that are coiling or engaged in an omega turn. We have also developed methods to distinguish dorsal from ventral bending and optimized the detection and quantification of parameters related to turns and reversals. To apply these methods, we have begun collecting new image data, with dorsal and ventral sides identified, on mutants previously defined as coiler Uncs. Currently, syd-1, unc-1, unc-10, unc-17, unc-26, unc-23, unc-37, and unc-75 are being classified and characterized for their movement such as body shape features, omega bends, reversals, speed, and coiling from collected data of 253 characteristics using an algorithm of Principle Component Analysis (PCA; Duda et al. 2001). The resultant cluster plot provides interesting insight into the relative similarities of specific coiler phenotypes and the behavioral profiles of mutants defective in different molecular pathways affecting nervous system function.

Zhaoyang Feng et al. (2003) International Worm Meeting "A relational database powered quantitative c. elegans behavioral analysis system and its possible application in systematic analysis of nicotinic signaling"

Zhaoyang Feng, William Schafer, Megan Palm, John Wittig

[International Worm Meeting, 2003]

C. elegans is particularly suitable for genetic analysis of the molecular basis of behavior because it has a simple nervous system of 302 neurons, and the synaptic connectivity, the cell line of each neuron are precisely known. Unfortunately, c. elegans behavior is one of most intractable biological phenomena: behavior changes from time to time; the behavioral differences among variants are subtle; and human observation is subjective and labor intensive. A quantitative c. elegans behavioral analysis system powered by relational database is developed in this study. Features extrapolated from machine vision analysis of c. elegans behavioral video are exported as two parts in database: the time to time behavioral data are saved in a widely available commercial spreadsheet format (Microsoft excel); and the statistical summary of time to time data is saved in a relational database. The features fall into three major catalogs: behavior (speed, reversal, turn angle, neck movement, etc), body posture (wave amplitude, wave length, etc), and morphology (body size, body length, etc). The outputted spreadsheet data is suitable for detail dissecting of several c. elegans behaviors including state-switch of locomotion. The system has been utilized in a study of the function of dopaminergic neurons in taping response, and a study of nicotinic signaling (Feng Z. et al, abstract in this meeting). Given the power of relational database, systematic analysis of c. elegans behavior is also possible. To test this possibility, the neural network feature selection, and principle component analysis (PCA) are utilized to study the effect of nicotine on N2 c. elegans, and several genetically engineered mutants. The features exported to database, and the systematic study results will be presented and discussed.