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[
International Worm Meeting,
2015]
Innate behaviors are hardwired into the nervous system, and are a response to either internal or external cues. The soil-dwelling nematode C. elegans uses a family of small molecules called "ascarosides" to signal a multitude of information about the physical and social environment [1]. Within these social cues are sex-specific behaviors, including hermaphrodite repulsion and male attraction [2]. We are interested in understanding the neural basis of male attraction to these cues. When exposed to the male-attractant ascaroside, ascaroside #8, male C. elegans show a prolonged dwell time within the chemical. This was tested using a two-drop choice assay, as developed previously [2]. This dwelling behavior is a result of neuromuscular changes within the worm in response to this cue. We hypothesized that these neuromuscular changes could be mediated by neuropeptides. Using a reverse genetic approach, we tested potential neuropeptides involved in ascaroside #8 meditated attraction. The largest family of neuropeptides in C. elegans, FMRFamide-like peptides (FLPs) are known to have neuromuscular effects [3], but a majority of FLPs still have uncharacterized phenotypes. Null mutants for FLPs expressed in male specific neurons were tested with ascaroside #8, and a decrease in attraction was found in multiple mutants. Having determined the necessity of FLPs for innate male attraction, via ascaroside#8, we are currently addressing the question of whether these neuromodulators are working synergistically at one site of action within the neural circuit or systematically throughout the entire animal.[1] Ludewig and Schroeder. Ascaroside signaling in C. elegans. www.wormbook.org[2] Srinivasan et. al. A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature. 2008.[3] Li and Kim. Family of FLP peptides in Caenorhabditis elegans and related nematodes. Frontiers in Endocrinology. 2014.
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[
International Worm Meeting,
2021]
Neuropeptides are small proteins produced in the nervous system that regulate synaptic communication by modulating the response of G-protein coupled receptor signals. Neuropeptides serve to repurpose neural circuits: a single gene can encode multiple peptidergic regulatory elements, making it difficult to identify roles of discrete and active neuropeptides. Here, we present a novel tool for the functional rescue neuropeptides that exploits an RNAi feeding-like technique using E. coli delivery to feed genetic loss-of-function C. elegans mutants active peptides. This technique allows us to tease apart unique roles of individual neuropeptides encoded by single genes. Using our rescue-by-feeding paradigm, we rescue three neuropeptides that have previously been rescued using canonical, transgenic methods -
pdf-1,
trh-1, and
ins-6 - to examine the efficacy of the paradigm. Our study confirms that some peptides are functionally redundant, while others that appeared to play similar roles in transgenic rescues are indeed unique. Finally, we argue that the mechanism of peptide delivery is reminiscent of the mRNA uptake observed in RNAi feeding paradigm, though we are currently exploring the mechanisms of how the peptide rescues the phenotype and altering delivery methods to work in other bacterivores. While these neuropeptide genes encode simpler pro-peptide products, we have recently employed this method to decipher the role of
flp-3-encoded peptides- a FMRFamide-like neuropeptide gene which encodes ten unique peptides. We postulate that this rescue-by-feeding paradigm can offer the ability to dissect the functional landscape of neuropeptide genes, previously inhibited by the bandwidth limitations of combinatorial transgenic analyses.
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[
International Worm Meeting,
2019]
The ability of organisms to communicate social information, often in the form of pheromones, is almost as ancient as chemical communication itself. However, there are still large gaps in our understanding as to how neurons transduce signals at a molecular level, and an even greater gap exists in understanding how sex-bias impacts neural networks and how sex impacts this form of inter-organismal communication. This study works to elucidate mechanisms involved in the sensation ascaroside #8 (ascr#8), a male-attractant pheromone utilized by C. elegans. Sensation of ascr#8 is solely dependent on the male-specific CEM neurons, which are present in both ventral-dorsal (V/D) and left-right (L/R) symmetries (CEM VL, VR, DL, DR). Single-cell transcriptomic analyses of these neurons identified two different G protein-coupled receptors (GPCRs) enriched in these cells;
srw-97, and
dmsr-12. Promoter-GFP expression analyses revealed that these receptors are expressed in specific, non-overlapping subsets of CEM neurons. Mutant worms deficient in either
srw-97 or
dmsr-12 display partial defects in ascr#8 response compared to wild-type worms. We hypothesized that removal of both these chemoreceptors will result in complete lack of attraction to ascr#8. We therefore generated a double mutant (
srw-97;
dmsr-12), and tested for response to ascr#8 using a single worm behavioral assay developed in our lab. This assay compares the attraction of individual male worms to ascr#8 to that over a vehicle control. The
srw-97;
dmsr-12 double mutants showed significant reduction in response to ascr#8. Characterization of these GPCRs as receptors for ascr#8 will increase our understanding of how sex-specific pheromone signaling is transduced.
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[
MicroPubl Biol,
2020]
We have previously described the expression patterns of all but one of the 102 homeobox genes present in the C. elegans genome, using either fosmid-based reporter transgenes or CRISPR/Cas9-engineered gfp reporter alleles (Reilly et al., 2020). The last remaining homeobox gene for which we were initially not able to generate a reporter reagent for is
ceh-84 (Reilly et al., 2020). As previously recognized by Thomas Brglin (Hench et al., 2015),
ceh-84 codes for an unusual homeodomain protein with two divergent homeodomains (Fig.1D). It likely is a tandem duplicate of the
ceh-85 gene, which has been classified as a pseudogene (Fig.1B).
ceh-84 has no recognizable homologs in other Caenorhabditis species (Hench et al., 2015). Together with its duplicate,
ceh-85, localizes to a large cluster of extensively duplicated genes on chromosome II, described by James Thomas (Thomas, 2006). Most of the duplicated genes code for MATH and BTB type proteins (Fig.1A). The entire cluster seems to be C. elegans-specific (Thomas, 2006) and, apart from a number of pseudogenes, it also contains the C. elegans-specific
ceh-81,
ceh-82 and
ceh-83 homeobox genes (Fig.1A).
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Kirshner A, Eddins D, French R, Helmcke K, Page GP, Linney E, Lnenicka G, Berger K, Welsh-Bohmer KA, Corl AB, Levin ED, Hirsch HV, Aschner M, Bartlett S, Possidente B, Hayden KM, Chen L, Possidente D, Ruden D, Heberlein U
[
Neurotoxicology,
2009]
Considerable progress has been made over the past couple of decades concerning the molecular bases of neurobehavioral function and dysfunction. The field of neurobehavioral genetics is becoming mature. Genetic factors contributing to neurologic diseases such as Alzheimer's disease have been found and evidence for genetic factors contributing to other diseases such as schizophrenia and autism are likely. This genetic approach can also benefit the field of behavioral neurotoxicology. It is clear that there is substantial heterogeneity of response with behavioral impairments resulting from neurotoxicants. Many factors contribute to differential sensitivity, but it is likely that genetic variability plays a prominent role. Important discoveries concerning genetics and behavioral neurotoxicity are being made on a broad front from work with invertebrate and piscine mutant models to classic mouse knockout models and human epidemiologic studies of polymorphisms. Discovering genetic factors of susceptibility to neurobehavioral toxicity not only helps identify those at special risk, it also advances our understanding of the mechanisms by which toxicants impair neurobehavioral function in the larger population. This symposium organized by Edward Levin and Annette Kirshner, brought together researchers from the laboratories of Michael Aschner, Douglas Ruden, Ulrike Heberlein, Edward Levin and Kathleen Welsh-Bohmer conducting studies with Caenorhabditis elegans, Drosophila, fish, rodents and humans studies to determine the role of genetic factors in susceptibility to behavioral impairment from neurotoxic exposure.
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Barrett, Alec, McWhirter, Rebecca, Vidal, Berta, Tavazoie, Saeed, Hobert, Oliver, Weinreb, Alexis, Miller, David, Xu, Chuan, Taylor, Seth, Paninski, Liam, Yemini, Eviatar, Sestan, Nenad, Basavaraju, Manasa, Litwin-Kumar, Ashok, Cros, Cyril, Reilly, Molly, Santpere, Gabriel, Poff, Abigail, Glenwinkel, Lori, Abrams, Alexander, Hammarlund, Marc, Rafi, Ibnul, Varol, Erdem, Oikonomou, Panos, Cook, Steven
[
International Worm Meeting,
2021]
There is strong prior evidence for genetic encoding of synaptogenesis, axon guidance, and synaptic pruning in neural circuits. Despite these foundational observations, the transcriptional codes that drive neural connectivity have not been elucidated. The C. elegans nervous system is a particularly useful model for studying the interplay between genetics and connectivity since its wiring diagram is highly stereotyped and uniquely well-defined by electron microscopy. Furthermore, recent evidence in C. elegans has suggested that a unique combination of transcription factors specifies each of the 118 neuron classes[1]. Motivated by evidence for the stereotypy of neural circuits and for the genetic encoding of neural identity, we introduce a novel statistical technique, termed Network Differential Gene expression analysis (nDGE), to test the hypotheses that neuron-specific gene expression dictates connectivity. Specifically, we test the hypothesis that pre-synaptic neural identity is defined by a "key" gene combination whose post-synaptic targets are determined by a "lock" gene combination. For our approach, we utilize neuron-specific gene expression profiles from the CeNGEN project[2] to investigate transcriptional codes for connectivity in the nerve ring[3]. We hypothesize that the expression of specific cell adhesion molecules (CAM) among synaptically-connected neurons drives synaptic maintenance in the mature nervous system. We posit that CAMs mediating synaptic stability would be more highly expressed in synaptically-connected neurons than in adjacent neurons with membrane contacts but no synapses. Thus, for each neuron, we compare the expression of all possible combinations of pairs of CAMs in the neuron and its synaptic partners relative to the neuron and its non-synaptic adjacent neurons. Two independent comparisons are generated, one for presynaptic neurons and a second result for postsynaptic neurons. Our nDGE analysis reveals that specific combinations of CAMs are correlated with connectivity in different subsets of neurons and thus provides a uniquely comprehensive road map for investigating the genetic blueprint for the nerve ring wiring diagram. Open source software of Network Differential Gene Expression (nDGE) is publicly available at https://github.com/cengenproject/connectivity_analysis along with a vignette showcasing the CAM results. 1. Reilly, M. B., Cros, C., Varol, E., Yemini, E., & Hobert, O. (2020). Unique homeobox codes delineate all the neuron classes of C. elegans. Nature, 584(7822), 595-601. 2. Taylor, S. R., Santpere, G., Weinreb, A., Barrett, A., Reilly, M. B., Xu, C. Varol, E., ... & Miller, D. M. (2020). Molecular topography of an entire nervous system. bioRxiv. 3. Cook, S. J.,... & Emmons, S. W. (2019). Whole-animal connectomes of both Caenorhabditis elegans sexes. Nature, 571(7763), 63-71.
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Barrett, Alec, Taylor, Seth R., Weinreb, Alexis, Miller III, David M., Varol, Erdem, Sestan, Nenad, Hobert, Oliver, McWhirter, Rebecca, Li, Mingfeng, Hammarlund, Marc
[
International Worm Meeting,
2021]
Advances in RNA-seq for bulk and single cell (sc) approaches have produced increasingly fine dissections of the C. elegans transcriptome. Although both techniques can yield transcriptomes for individual cell types, each comes with strengths and weaknesses. scRNA-Seq affords high resolution, but suffers from dropout, leading to false negatives. Bulk sequencing detects more genes, but suffers from contaminating cell types, resulting in false positives. In this work we integrated these orthogonal approaches to improve the accuracy of both methods. We used bulk samples collected for specific neuron types and sc datasets for all C. elegans neurons and additional non-neuronal cells (1). We used sc data to estimate contamination in each bulk sample, and developed novel methods for removing these gene counts. In one approach we used linear histogram matching to scale sc counts, and subtracted putative contamination using data from non-neuronal clusters. In another approach we used bootstrapping to estimate gene level contributions from target and contaminating tissues in sc data and apply them to bulk counts, providing a bootstrap sample distribution of corrected expression data. We assessed these approaches in two ways: 1) Measuring improvements in calling genes with known expression in all neurons; 2) Examining effects on eliminating genes expressed exclusively in contaminating tissues. We found that our analysis reduced false positives, while maintaining robust true positive detection, thus offering a unique strategy for utilizing complementary bulk and sc RNA-Seq data sets to enhance the accuracy of cell-specific expression profiling data. 1. Taylor SR, Santpere G, Weinreb A, Barrett A, Reilly MB, Xu C, et al. Molecular topography of an entire nervous system. bioRxiv. 2020:2020.12.15.422897.
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[
International Worm Meeting,
2021]
The conserved mitogen activated kinase kinase kinase (MAPKKK) DLKs play critical roles in neuronal process growth, synapse formation and axon regeneration. DLKs also act as sensors to mediate neuronal responses to different stresses, including microtubule (MT) disruption. However, it is not clear in which contexts DLKs sense MT stress and which signaling transduction mechanisms control the activity of DLKs to mediate specific functional outcomes. In C. elegans the DLK ortholog DLK-1 is normally expressed in most neurons at low levels. From a forward genetic screen for mutants displaying aberrant DLK-1 levels or localization, we identified a mutant that exhibited aggregated DLK-1 in neuronal soma. Using whole genome sequencing and genetic mapping, we determined that this mutation affected
ben-1, one of six beta-tubulin isotypes in C. elegans. Levels of the transcription factor CEBP-1, a downstream target induced by DLK-1, were elevated in neurons of
ben-1 mutants. These findings suggest that altering
ben-1 function activates
dlk-1, resulting in
cebp-1 dependent transcription. A
ben-1 transcriptional reporter is exclusively expressed in neurons. BEN-1 is known to convey the sensitivity to benomyl1, a microtubule-targeting compound used for killing parasitic nematodes and treating cancers. We found that benomyl treatment also caused neural DLK-1 aggregation, CEBP-1 elevation, as well as neuronal defects such as impaired synaptogenesis. Altogether, our findings suggest that changes in neuronal beta-tubulin BEN-1 containing MT cytoskeleton elicit specific stress responses to activate
dlk-1/cebp-1 signaling. 1. Driscoll, M., Dean, E., Reilly, E., Bergholz, E., and Chalfie, M. (1989). Genetic and molecular analysis of a Caenorhabditis elegans beta-tubulin that conveys benzimidazole sensitivity. J. Cell Biol. 109, 2993-3003.
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[
International Worm Meeting,
2019]
The pharyngeal nervous system is composed of only 20 neurons belonging to 14 different types, which form a self-contained circuit that is almost independent from the somatic nervous system. This simplicity makes it possible to analyze it in a comprehensive way. Moreover, all pharyngeal neurons directly connect to end organs and can be considered polymodal with sensory-motor characteristics (see abstract by S.J. Cook and S.W.Emmons), a feature that is reminiscent of primitive nervous systems. Thus, understanding how pharyngeal neurons are specified during development might shed light on fundamental aspects of neuronal development.
ceh-34, a homeodomain transcription factor of the Six family, is continuously expressed in all pharyngeal neurons and no other neurons outside of the pharynx. Remarkably, we have found that in
ceh-34 mutants, pharyngeal neurons are generated, but fail to express a wide array of terminal identity genes, including neurotransmitter pathway genes, indicating that
ceh-34 acts as a pharyngeal neuron master regulator ("terminal selector"). Moreover, a conditional AID-based allele demonstrates that
ceh-34 is continuously required during the life of the worm to maintain pharyngeal neuron identity. We hypothesize that
ceh-34 acts together with other transcription factors to form a combinatorial code that gives each pharyngeal neuron its unique identity. We have found several other homeodomain transcription factors expressed in subsets of pharyngeal neurons (see abstract by M. Reilly and O.Hobert) and we are doing a mutant analysis to test whether they also play a role in the pharyngeal nervous system specification. Moreover, we are performing forward genetic screens to find additional factors in an unbiased way. So far we have identified four mutants that appear to affect I2 neuron identity. We are in the process of pinpointing the causal molecular lesions and further characterizing these new mutants. We hope our efforts will lead to a comprehensive understanding of the regulatory code that dictates pharyngeal neuron development.
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[
J Vis Exp,
2010]
American Biologist Martin Chalfie shared the 2008 Nobel Prize in Chemistry with Roger Tsien and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Martin Chalfie was born in Chicago in 1947 and grew up in Skokie Illinois. Although he had an interest in science from a young age--learning the names of the planets and reading books about dinosaurs--his journey to a career in biological science was circuitous. In high school, Chalfie enjoyed his AP Chemistry course, but his other science courses did not make much of an impression on him, and he began his undergraduate studies at Harvard uncertain of what he wanted to study. Eventually he did choose to major in Biochemistry, and during the summer between his sophomore and junior years, he joined Klaus Weber's lab and began his first real research project, studying the active site of the enzyme aspartate transcarbamylase. Unfortunately, none of the experiments he performed in Weber's lab worked, and Chalfie came to the conclusion that research was not for him. Following graduation in 1969, he was hired as a teacher Hamden Hall Country Day School in Connecticut where he taught high school chemistry, algebra, and social sciences for 2 years. After his first year of teaching, he decided to give research another try. He took a summer job in Jose Zadunaisky's lab at Yale, studying chloride transport in the frog retina. Chalfie enjoyed this experience a great deal, and having gained confidence in his own scientific abilities, he applied to graduate school at Harvard, where he joined the Physiology department in 1972 and studied norepinephrine synthesis and secretion under Bob Pearlman. His interest in working on C. elegans led him to post doc with Sydney Brenner, at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England. In 1982 he was offered position at Columbia University. When Chalfie first heard about GFP at a research seminar given by Paul Brehm in 1989, his lab was studying genes involved in the development and function of touch-sensitive cells in C. elegans. He immediately became very excited about the idea of expressing the fluorescent protein in the nematode, hoping to figure out where the genes were expressed in the live organism. At the time, all methods of examining localization, such as antibody staining or in situ hybridization, required fixation of the tissue or cells, revealing the location of proteins only at fixed points in time. In September 1992, after obtaining GFP DNA from Douglas Prasher, Chalfie asked his rotation student, Ghia Euskirchen to express GFP in E. coli, unaware that several other labs were also trying to express the protein, without success. Chalfie and Euskirchen used PCR to amplify only the coding sequence of GFP, which they placed in an expression vector and expressed in E.coli. Because of her engineering background, Euskirchen knew that the microscope in the Chalfie lab was not good enough to use for this type of experiment, so she captured images of green bacteria using the microscope from her former engineering lab. This work demonstrated that GFP fluorescence requires no component other than GFP itself. In fact, the difficulty that other labs had encountered stemmed from their use of restriction enzyme digestions for subcloning, which brought along an extra sequence that prevented GFP's fluorescent expression. Following Euskirchen's successful expression in E. coli, Chalfie's technician Yuan Tu went on to express GFP in C. elegans, and Chalfie published the findings in Science in 1994. Through the study of C. elegans and GFP, Chalfie feels there is an important lesson to be learned about the importance basic research. Though there has been a recent push for clinically-relevant or patent-producing (translational) research, Chalfie warns that taking this approach alone is a mistake, given how "woefully little" we know about biology. He points out the vast expanse of the unknowns in biology, noting that important discoveries such as GFP are very frequently made through basic research using a diverse set of model organisms. Indeed, the study of GFP bioluminescence did not originally have a direct application to human health. Our understanding of it, however, has led to a wide array of clinically-relevant discoveries and developments. Chalfie believes we should not limit ourselves: "We should be a little freer and investigate things in different directions, and be a little bit awed by what we're going to find."