Kim, B. et al. (2017) International Worm Meeting "A neural cell adhesion protein code for nervous system connectivity."

Emmons, S.W., Kim, B., Ivashkiv, O.

[International Worm Meeting, 2017]

Most synaptic connections in animal nervous systems are genetically specified. How this genetic encoding is brought about is largely unknown. We are addressing this question by focusing on the fully described neural circuits in the C. elegans male tail, which subserve copulation. The molecules thought to act as cell-recognition tags for synaptic specificity comprise a largely evolutionarily conserved set of transmembrane or secreted proteins with extracellular protein-protein interaction domains, such as IG, LRR, EGF, cadherin, fibronectin, and so forth. There are 106 such proteins (cell adhesion proteins) encoded in the C. elegans genome. We hypothesize that there must be a correlation between the expression of these genes and connectivity. To find such a correlation, using reporter genes we have examined almost the entire set of 106 genes in the 238 neurons and muscles in the male tail neural network. Seventy-seven of the 98 genes tested so far (79%) are expressed. The expression matrix is complex. Most genes are expressed in multiple, unrelated cells and muscles, while most cells and muscles express multiple genes (up to 27 in bodywall muscles). This arrangement is consistent with an abstract combinatorial code for synaptic connectivity. We now seek statistical correlations of individual genes or combinations of genes with connectivity that we hope will reveal the nature of the code. We have validated the use of expression data for identifying genes critical for connectivity by experimentally verifying the roles of 7 genes in formation of a specific set of connections in the male tail (see abstract by Kim and Emmons).

Kim, B. et al. (2017) International Worm Meeting "Conserved cell adhesion protein interactions mediate neural wiring of a sensory circuit in the C. elegans male."

Kim, B., Emmons, S.W.

[International Worm Meeting, 2017]

The function of the nervous system relies on precise synaptic connections. A number of cell adhesion proteins are implicated in the cell recognition of synaptic partners, but how these proteins determine the synaptic connectivity in vivo is poorly understood. Here, we show that two pairs of interacting cell adhesion proteins regulate the neural wiring of a male-specific sensory circuit in C. elegans. This circuit is generated during the L4-adult transition: a pair of sex-shared PHC neurons extends processes that follow and make synapses with the axon of the sex-shared interneuron AVG; later, a male-specific sensory neuron HOA adds its axon to the PHC-AVG bundle, making a circuit composed of three neurons with extensive synaptic connections. Using expression, mutant analysis, cell-specific rescue experiments, and protein binding studies, we identify CASY-1/calsyntenin and RIG-6/contactin as postsynaptic factors acting in AVG for axon fasciculation between synaptic partners (see abstract by Kim, Ivashkiv, and Emmons). In the presynaptic cells, BAM-2/neurexin-related is expressed in HOA and is a binding partner of CASY-1 while SAX-7/L1CAM is expressed in PHC and is a binding partner of RIG-6. Axon fasciculation defects of casy-1 and rig-6 mutants are distinguishable: in mutants lacking casy-1, the HOA axon was frequently detached from the PHC-AVG axon bundle, whereas in rig-6 mutants, both HOA and PHC axons were detached from AVG, but the HOA-PHC fasciculation was intact. Cell ablation studies revealed that HOA-AVG axon fasciculation is dependent on PHC, raising the possibility that in rig-6 mutants the HOA axon may follow the PHC process separated from AVG. Structure-function analyses reveal that the LNS (Laminin, Neurexin, Sex hormone-binding globulin) domain, but not the cadherin domains, of CASY-1 and the immunoglobulin domains of RIG-6 are responsible for the axon fasciculation. Mutations in all of these cell adhesion proteins disrupt male vulva location behavior during mating, consistent with the functions of these neurons. Our findings suggest that cell-cell recognition via phylogenetically conserved protein interactions of neural cell adhesion proteins is a crucial component of neural circuit assembly.

Fluet A et al. (1998) West Coast Worm Meeting "AGGREGATION AND TOXICITY OF HUMAN b-AMYLOID PEPTIDE VARIANTS EXPRESSED IN C. ELEGANS"

Fluet A, Johnson CJ, Fay DS, Link CD

[West Coast Worm Meeting, 1998]

We have engineered transgenic C. elegans that express the human b-amyloid peptide, which appears to play a central role in the pathology of Alzheimer's disease. As described previously, these worms, which express the peptide under the control of the unc-54 promoter, are unhealthy, have larval vacuoles, and exhibit a progressive paralysis. To better understand the molecular mechanism of b peptide toxicity, we would like to determine which, if any, of these phenotypes are specific to the expression of b peptide, and which are non-specific effects resulting from overexpression of a foreign peptide. To address this question we have sought to develop non-toxic b peptide variants, which should 'unmask' the non-specific phenotypes. We have specifically attempted to design b peptide variants that are incapable of aggregating into amyloid fibers, which have previously been reported to be directly correlated with b peptide toxicity. Single amino acid changes in the b(1-42) peptide have provided us with two interesting variants that both fail to produce amyloid aggregates: L17P and M35C. Intriguingly, multiple L17P transgenic lines all show the progressive paralysis observed in strains expressing wild-type b peptide. All M35C lines, however, are significantly healthier than the strains expressing wild-type b peptide. While the L17P variant may allow us to differentiate between b peptide's ability to form aggregates and its toxicity, the M35C variant is currently being used to differentiate between specific and non-specific toxicity resulting from b peptide expression. We have also isolated a second non-aggregating version of the b peptide, the single-chain b dimer. Like the M35C variant, this version of the b peptide does not aggregate and animals that express it are healthier than those expressing the wild-type b peptide. Quantitative immunoblot analyses on these strains demonstrate that the differences we see in aggregation of the variants are due to qualitative differences in the peptides and not quantitative differences in their levels of expression. These results also suggest that the progressive paralysis phenotype is specifically due to b peptide toxicity. We are currently using the non- or less-toxic b peptide variants in conjunction with the wild-type b peptide to look for molecular correlates of b peptide toxicity. We have preliminary results from immunoblots of Hsp16 expression that suggest that this heat shock protein is upregulated in response to b amyloid expression, and that this upregulation is significantly reduced in lines expressing the non-aggregating b dimer.

Sakai, N. et al. (2019) International Worm Meeting "Roles of cell adhesion proteins in the C. elegans male tail."

Sun, P., Kim, B., Sakai, N., Hasan, R., Emmons, S.

[International Worm Meeting, 2019]

Our lab has described the complete neuronal connectivity (Jarrel et al., 2012) and the expression patterns of 100 neural cell adhesion genes (unpublished data) across 173 neurons and 64 muscles in the C. elegansmale tail. This gives us the opportunity to address the relationship between the cell adhesion proteins and male tail development. We found that A-type and B-type ray neurons, derived from a single ray precursor cell, express non-overlapping sets of cell adhesion genes. To address whether this neuron-type-specific expression determines ray development, we are analyzing ray morphology and ray neuron connectivity in cell adhesion gene mutants. We visualize RnB presynapses using a RnB-specific promoter (pkd?2) driving expression of fluorescence-tagged, pre-synaptic protein RAB?3, and connectivity to EF interneurons using iBLINC. Mutations in 6 of the 8 expressed cell adhesion proteins have been tested so far (cdh-12, fmi-1, hmr-1, nrx-1 lron-7, rig-3), as well as in one gene expressed in EF (nlg-1). The only observed effect has been a slightly increased rab-3 level and iBLINC signal in nrx-1(-). We have observed no defects in ray morphology. Previously (Baird et al., 1991), we postulated that transcription factors controlling ray identities, mutations in which caused fused rays and additional changes such as in neurotransmitter expression, acted in part by controlling the expression of cell adhesion genes. Accordingly, we are now analyzing the expression of ray cell adhesion genes in mab-5, egl-5, mab-18,and mab-21 mutants.

Kim, Do-Young et al. (2019) International Worm Meeting "FMRFamide-related neuropeptide FLP-12 regulates head locomotion of C. elegans."

Li, Chris, Kim, Do-Young, Kim, Jinmahn, Park, Cheon-Gyu, Suh, Byung-Chang, Kim, Kyuhyung

[International Worm Meeting, 2019]

Neuropeptides regulate a multitude of behaviors in many organisms (Li et al., 2014). However, mechanisms by which functions of neuropeptides are coordinated to generate proper behaviors are not fully understood. In C. elegans, the SMB motor neurons consist of two pair of neurons that innervate head/neck muscles (White et al, 1986) and shape the amplitude of sinusoidal movement (Gray et al, 2005, Kim et al., 2014), indicating a role of SMB in head locomotion. The SMB neurons have shown to express FMRFamide-related neuropeptide FLP-12 (Kim and Li, 2004, Kim et al., 2014). To identify roles of FLP-12 neuropeptide in head locomotive behavior, we first observed phenotypic consequences by loss of FLP-12. We measured five locomotive parameters including omega turn, reversal, head lift, foraging and frequency of head movement. We found that all five locomotive parameters are increased in flp-12 mutants. We fully rescued defects in head locomotion of flp-12 mutants by expressing flp-12cDNA specifically in SMB. These results indicate that FLP-12 may act in the SMB to regulate head movement. To identify receptors for FLP-12, we performed RNAi screening against a total of 16 putative neuropeptide receptor genes. We found that knock-down of several genes including frpr-8 exhibits increases foraging behavior similarly to flp-12 mutants and frpr-8 mutants. Expressing frpr-8 cDNA with its own promoter successfully rescues foraging phenotype of frpr-8. Heterogously expressed FRPR-8 in HEK cells are sufficient to confer Ca2+ response upon FLP-12 exposure. Altogether, our data expand our understanding of how a single neuropeptide and its receptor modulate behaviors.

Emmons, S.W. et al. (2019) International Worm Meeting "The molecular code for neural connectivity."

Bloniarz, A.E., Kim, B., Emmons, S.W., Sakai, N.

[International Worm Meeting, 2019]

A large number of conserved cell adhesion proteins have been implicated in synapse formation, suggesting great molecular complexity at the synapse. Conclusions about functions are often based on effects at particular cell contacts or synapses, for example repulsion, or induction of pre- or post-, excitatory or inhibitory synapse formation. We suggest, on the contrary, that these proteins are neutral molecular tags for cell recognition; their complex expression in the nervous system reflects their role as the code for connectivity. We have determined comprehensively the expression of cell adhesion genes in a fully described C. elegans neural network - the 173 neuron, 64 muscle network that governs male mating - based on the premise that it should be possible to find a correlation between expression of these genes and connectivity. Eighty percent of the 100 cell adhesion genes tested are expressed in the network. The pattern is one expected for an abstract, combinatorial code: each gene is expressed in multiple, unrelated neurons and muscles, while most cells express multiple genes. Altogether, taken over the 3209 chemical and gap junction edges in the connectivity matrix, there are 60,894 instances of possible pairings of genes in cells connected by synapses, illustrating the complexity. The most frequently used genes include sax?3/ROBO; sax?7/L1CAM; nrx?1/neurexin, syg?1, and many other well-studied genes. There have been two main thrusts of the statistical analyses so far: looking for surprising properties of the subset of neurons and muscles expressing a gene or sets of genes, and fitting models that predict connectivity from expression. For a subset of genes, we found evidence for homophily from a greater amount of connectivity among co-expressing cells compared to expectation in a null distribution of connectivity of random subcircuits. We use machine learning methods to fit predictive models of connectivity given the set of cell adhesion protein expression tags, and assess their accuracy on a test set of neurons. Such an approach ? predicting connectivity from cell adhesion gene expression ? might provide a route to the connectomes of nervous systems more feasible than EM reconstruction.

Xu, T. et al. (2019) International Worm Meeting "How does an animal move? An integrative model of C. elegans forward locomotion."

Kawano, T., Xu, T., Po, M., Shao, S., Wu, M., Zhen, M., Huo, J., Wen, Q., Lu, Y.

[International Worm Meeting, 2019]

Descending signals from the brain play critical roles in controlling and modulating locomotion kinematics. In the Caenorhabditis elegans nervous system, descending AVB premotor interneurons exclusively form gap junctions with the B-type motor neurons that execute forward locomotion. We combined genetic analysis, optogenetic manipulation, calcium imaging, and computational modeling to elucidate the function of AVB-B gap junctions during forward locomotion. First, we found that some B-type motor neurons generate rhythmic activity, constituting distributed oscillators. Second, AVB premotor interneurons use their electric inputs to drive bifurcation of B-type motor neuron dynamics, triggering their transition from stationary to oscillatory activity. Third, proprioceptive couplings between neighboring B-type motor neurons entrain the frequency of body oscillators, forcing coherent bending wave propagation. Despite substantial anatomical differences between the motor circuits of C. elegans and higher model organisms, converging principles govern coordinated locomotion.

Melissa Harrison et al. (2001) International C. elegans Meeting "FUNCTIONAL STUDIES OF THE CLASS B SYNMUVS REQUIRED FOR THE NEGATIVE REGULATION OF VULVAL INDUCTION AND CHARACTERIZATION OF A NOVEL CLASS B SYNMUV GENE"

Melissa Harrison, Bob Horvitz

[International C. elegans Meeting, 2001]

A receptor tyrosine kinase/Ras pathway necessary for vulval induction in C. elegans has been shown to be negatively regulated by two redundant pathways, defined by the synthetic Multivulva (synMuv) class A and B genes. Mutations in members of either class alone do not result in a Multivulva phenotype. However, animals containing loss-of-function mutations in both a class A and a class B gene display a synMuv phenotype. While most of the identified class A genes encode novel proteins, many of the class B synMuv genes, including lin-35 Rb, dpl-1 DP, efl-1 E2F, lin-53 RbAp48, hda-1 HDAC, and let-418 Mi-2, have homologs in other organisms known to be involved in chromatin modification complexes. Three mutations that define a new class B synMuv were identified in a recent screen for additional class B genes performed with Craig Ceol and Frank Stegmeier in our laboratory. We mapped these mutations to the far left of the X chromosome and are currently attempting to clone the gene by cosmid rescue. Several mammalian homologs of the class B synMuv genes have been shown to be components of the NuRD chromatin remodelling complex. Additionally, some of the proteins in the synMuv B pathway have been shown to physically interact either in vitro (1,2) or in yeast two-hybrid assays (3). We are attempting to use co-immunoprecipitation experiments to expand the current knowledge of the presumptive class B protein complex in C. elegans . Analysis of the effects of various mutations on co-immunoprecipitation may allow us to assign functionality to some novel class B genes. Currently, there is little understanding of how the class A and B synMuvs redundantly regulate vulval induction. Class A and B synMuvs might play redundant roles in the formation of a chromatin modifying complex, and such physical interactions could also be demonstrated through co-immunoprecipitaion experiments. (1) Ceol, C. and H.R. Horvitz (in press). (2) Lu and Horvitz. (1998) Cell 95 : 981. (3) Walhout et al. (2000) Science 287 :116.

Sun, Simo et al. (2017) International Worm Meeting "Neural and molecular basis involved in low preference to Bifidobacterium infantis in Caenorhabditis elegans."

Nishikawa, Yoshikazu, Kage-Nakadai, Eriko, Sun, Simo

[International Worm Meeting, 2017]

Neural and molecular mechanisms underlying food preference have been poorly understood. We previously showed that Bifidobacterium infantis, one of the well-known probiotic bacteria, extend the lifespan of Caenorhabditis elegans (Ikeda et al., Appl. Environ. Microbiol., 2007). In this study, we found that C. elegans exhibited low preference to B. infantis when compared with a standard food E. coli. Genetic screens identified tol-1 (a sole homolog of mammalian Toll-like receptors) and daf-16/FoxO as positive regulators of low preference to B. infantis. Worms with mutations in canonical TLR signaling molecules (pmk-3, mom-4 and ikb-1) that are employed in avoidance behavior to a pathogenic bacterium Serratia marcescens (Brand et al.,Curr. Biol., 2015) showed a normal behavior to B. infantis, suggesting alternative mechanisms. Genetic analyses revealed that the function of daf-16 isoform b in AIY neurons is responsible for the behavior to B. infantis. Because daf-16b regulates the AIY neurons morphology during development (Christensen et al., Development, 2011), daf-16b may play roles in AIY development to organize low preference to B. infantis.

AI Cid et al. (1999) International C. elegans Meeting "Investigating the function of the SMA-1 SH3 domain in C. elegans morphogenesis"

AI Cid, J Austin

[International C. elegans Meeting, 1999]

Spectrin is a cytoskeletal actin binding protein made up of two subunits each of the proteins a -spectrin and b -spectrin. We have identified an unusual member of the b -spectrin family, SMA-1, that is required for proper morphogenesis of the C. elegans embryo. SMA-1 is a homolog of Drosophila b H -spectrin. Like other b -spectrins, SMA-1 and b H -spectrin contain an N-terminal actin binding domain, a series of spectrin repeats and a C-terminal pleckstrin homology domain. However, SMA-1 and b H -spectrin contain more spectrin repeats than other b -spectrins, and have an SH3 domain not found in other b -spectrins. SMA-1 and b H -spectrin also differ from other b -spectrins in their localization within the cell: b -spectrin is found laterally, while SMA-1 and b H -spectrin are found apically. SH3 domains are protein-protein interaction motifs which bind ligands containing proline-rich regions. We would like to know the function of this domain within SMA-1. The SMA-1 SH3 domain may play a number of roles in SMA-1 function: it could be responsible for SMA-1 localization within the cell, or it could interact with proteins that regulate SMA-1 function. We would like to determine if the SMA-1 SH3 domain plays a role in SMA-1's intracellular localization or more directly in its function in morphogenesis. We are using a yeast two-hybrid screen to identify SMA-1 SH3 domain ligands. We have identified several positives from our screen which we are in the process of sequencing. We also plan to do more in depth analysis of the SMA-1 SH3 domain, making specific mutations in the SMA-1 SH3 and examining loss-of-function phenotypes of potential SMA-1 SH3 ligands by RNA interference. To address whether the SH3 domain is responsible for SMA-1 protein localization we have begun to make GFP protein fusions containing the SMA-1 SH3 domain. By transforming worms with these SH3-GFP fusion constructs we will be able to determine where the SMA-1 SH3 domain localizes within the cell. With these methods we can attempt to understand what role the SMA-1 SH3 plays in C. elegans morphogenesis.