Palumbos, Sierra et al. (2021) International Worm Meeting "cAMP controls a trafficking mechanism that directs the neuron specificity and subcellular placement of electrical synapses"

Palumbos, Sierra, McWhirter, Rebecca, Von Stetina, Steve, Miller III, David M, Skelton, Rachel

[International Worm Meeting, 2021]

Despite the functional importance of electrical synapses, the molecular mechanisms that direct the formation of neuron-specific gap junctions remain largely unknown. To address this question, we identified targets of the UNC-4 transcription factor that controls connectivity in the C. elegans motor circuit. UNC-4 functions in VA motor neurons to direct the formation of gap junctions on the VA axon with the interneuron AVA (VA-AVA). Locomotion is disrupted in unc-4 mutants because VAs are miswired with electrical input from the interneuron AVB (VA-AVB) which aberrantly form on the VA soma. Thus, UNC-4 controls both the specificity and subcellular placement of electrical synapses. We determined that UNC-4 blocks expression of two antagonists of cAMP, the phosphodiesterase, PDE-1, and the Go/Gi-coupled GPCR, FRPR-17, to prevent assembly of ectopic VA-AVB gap junctions. This finding suggests that cAMP signaling promotes the formation of functional wild-type VA-AVA gap junctions. We validated this hypothesis by showing that optogenetic elevation of cAMP rescues the Unc-4 movement defect and thus restores VA-AVA circuit function. In addition, forced depletion of cAMP in VAs phenocopies unc-4 mutants. Because gap junction placement is shifted from the VA axon to cell soma in unc-4 mutants, we reasoned that trafficking of gap junction components could be perturbed. Live-cell imaging of the gap junction protein, GFP-UNC-9, confirmed that trafficking into the VA axon is strikingly impaired in unc-4 mutants. Genetic activation of cAMP signaling is sufficient to restore GFP-UNC-9 trafficking in VAs. Thus, we propose that cAMP directs both the specificity and placement of electrical synapses by activating mechanisms that transport gap junction components into the VA axonal compartment. Although studies in cultured cells have implicated cAMP in gap junction assembly, our in vivo experiments now firmly establish that cAMP regulates the biogenesis of electrical synapses in an intact nervous system.

Byrne, Alexandra B. et al. (2015) International Worm Meeting "PARGs and PARPs: Novel Regulators of Axon Regeneration."

Miller, III, David M., Hammarlund, Marc, McWhirter, Rebecca D., Byrne, Alexandra B.

[International Worm Meeting, 2015]

Few of the intrinsic mechanisms that regulate axon regeneration after injury are known. To identify genes that regulate axon regeneration, we compared gene expression profiles of FACS-sorted C. elegans GABA motor neurons with high regenerative capacity (conferred by overexpression of DLK-1 MAPKKK) to wild type GABA motor neurons. We detected robust upregulation of both poly(ADP-ribose) glycohydrolases (PARGs), pme-3 and pme-4, in neurons with high regenerative capacity. These data suggest PARG activity might promote axon regeneration. We performed laser axotomy in pme-3 and pme-4 loss-of-function mutants and found that regeneration is impaired. Therefore, PARGs are regeneration-promoting factors.PARGs degrade poly(ADP-ribose), which is synthesized by poly(ADP-ribose) polymerases (PARPs). Thus, the balance between PARG and PARP activity determines cellular levels of poly(ADP-ribose). The PARG-PARP balance regulates multiple processes including DNA damage response, lifespan, and neurodegeneration. Since PARGs counteract PARP function, we hypothesized that loss of PARP activity would have the opposite effect on axon regeneration to loss of PARG activity. We found that loss of function of PARP genes pme-1 and pme-2 increased axon regeneration. Therefore, PARPs inhibit axon regeneration. Together with our PARG findings, these data suggest that levels of poly(ADP-ribose) are a critical determinant of regenerative potential.Next, we investigated whether we could inhibit PARP activity post-injury to promote regeneration of damaged axons. Multiple PARP inhibitors are currently in clinical trials for indications including cancer and stroke. We tested whether PARP inhibitors could enhance axon regeneration. Wild type animals treated with chemical PARP inhibitors after injury showed significantly enhanced axon regeneration compared to controls. Thus, PARP activity regulates the acute response of neurons to axon injury, and PARP inhibition after injury is sufficient to improve regeneration. Together, our findings identify a novel pathway involving control of poly(ADP-ribose) levels that regulates axon regeneration.

He, Siwei et al. (2015) International Worm Meeting "Transcriptional control of postsynaptic remodeling through regulated expression of an immunoglobulin superfamily protein."

Taub, Daniel, Francis, Michael, Philbrook, Alison, McWhirter, Rebecca, Miller, David, Gabel, Christopher, He, Siwei

[International Worm Meeting, 2015]

Neural circuits are extensively remodeled during development; however, the mechanisms underlying this process and the timing of rewiring remain largely unknown. Here we describe a transcriptionally regulated immunoglobulin super family protein, OIG-1, that fine-tunes synaptic plasticity in remodeling GABAergic motor neurons. DD class GABAergic motor neurons reverse polarity in the first larval (L1) stage. In newly born animals, DDs receive cholinergic inputs in the dorsal nerve cord but these are switched to the ventral side by the end of the L1 stage. The relocation of the DD postsynaptic apparatus can be monitored with the acetylcholine receptor (AChR) subunit, ACR-12::GFP. OIG-1 is highly expressed in early DD neurons where it antagonizes the relocation of ACR-12::GFP from the dorsal side. During the L1/L2 transition, OIG-1 is down-regulated by the transcription factor, IRX-1/Iroquois, in DD neurons to coincide with the translocation of postsynaptic ACR-12 to the ventral side. In VD class GABAergic motor neurons, which normally do not remodel, the transcription factor, UNC-55/COUP-TF turns off IRX-1, thus maintaining high levels of OIG-1 to block the removal of dorsally-located ACR-12 receptors. OIG-1 is secreted from GABAergic motor neurons but its anti-plasticity function is cell-autonomous and does not require secretion. Instead, we propose that secretion offers a rapid mechanism for clearing functionally active, intracellular OIG-1 thereby unleashing the postsynaptic remodeling program. Our study provides a novel mechanism by which synaptic remodeling is set in motion, through the temporal and spatial regulation of an Ig domain protein. .

Kroetz, Mary B. et al. (2011) International Worm Meeting "Sex-specific regulation of gonadal development in C. elegans."

McWhirter, Rebecca D., Spencer, W. Clay, Zarkower, David, Miller, III, David M., Berkseth, Matthew R., Kroetz, Mary B.

[International Worm Meeting, 2011]

The gonad of C. elegans originates as a four-cell primordium in the embryo. Initially it is morphologically identical in both sexes, but soon after the animal completes embryogenesis the gonad begins to develop into one of two distinct sex-specific organ structures. Despite the extensive sexual dimorphism and previously defined cell lineages of the gonad, the genetic pathways that direct the sex-specific development of this organ remain largely unknown. All sexual dimorphism is under the control of the TRA-1 transcriptional regulator, but no gonadal direct targets of TRA-1 are known. The overall aim of this work is to define the genetic network of sex-specific gonadal development, focusing on the direct targets of TRA-1. To identify the sex-specific gonadal regulators, cell-specific microarray-based mRNA expression profiling was conducted. Single sex populations of animals were generated by employing sex determination pathway mutants. The somatic gonadal precursor cells (Z1/Z4) expressed a gfp reporter allowing for their identification by FACS from dissociated embryonic cells. Z1/Z4-enriched transcripts were identified for both sexes. The majority of proteins with known Z1/Z4-enriched expression were identified by this method, and a number of novel Z1/Z4-enriched transcripts have subsequently been validated by reporter analysis, confirming the effectiveness of this approach. Work is currently underway to validate several of the sex-enriched transcripts. Recently our laboratory has conducted TRA-1 chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Comparison of the sex-specific gonadal transcriptome with the direct targets of TRA-1 as identified by ChIP-seq should identify which sex-specific transcripts are directly regulated by TRA-1 and presumably at the top of the hierarchy of gonadal regulators.

Spencer, William Clay et al. (2009) International Worm Meeting "Genomic Strategies to Map the C. elegans Transcriptome."

Watkins, Kathie, Spencer, William Clay, Gerstein, Mark, McWhirter, Rebecca, Miller III, David, Watson, Joseph, Kern, Nurith, Agarwal, Ashish, Wang, Shenglong

[International Worm Meeting, 2009]

The C. elegans genome is completely sequenced yet many predicted genes lack biological evidence for transcription. Additionally, a substantial number of cryptic protein-coding genes and ncRNAs (miRNAs, snoRNAs, etc.) are likely to have been overlooked by gene prediction software. To identify these transcripts, we are isolating RNA from specific C. elegans cells and tissues for tiling array analysis and for high throughput cDNA sequencing (RNA-Seq). This approach ensures detection of rare RNAs from small populations of cells while also providing clues to their in vivo functions. Our data sets will be merged with complementary results from other laboratories in the modENCODE consortium (modENCODE.org) to provide a detailed picture of the C. elegans transcriptome. We use specific promoters to mark cells for isolation by FACS or for mRNA extraction by the mRNA tagging method. The small amount of RNA obtained by these methods (<25 ng) is amplified to generate a labeled ds cDNA target for hybridization to the Affymetrix C. elegans Tiling 1.0R array. To date, we have generated tiling array profiles of >20 different cells and tissues including neurons, muscle, intestine, hypodermis, excretory cell, coelomocytes, etc. Threshold analysis detects transcripts from established gene models as well as from candidate transcriptionally active regions (TARs) in intergenic and intronic domains. Biased detection of known tissue and cell-specific transcripts validates these data sets and suggests that other differentially expressed TARs may exercise cell-specific functions. In addition to detecting novel transcripts, our approach is expected to produce gene expression maps that match the single cell resolution of the C. elegans anatomy.

Barrett, Alec et al. (2021) International Worm Meeting "Integrating bulk and single cell transcriptomics for accurate detection of tissue-specific gene expression"

Li, Mingfeng, Barrett, Alec, Weinreb, Alexis, Hammarlund, Marc, McWhirter, Rebecca, Hobert, Oliver, Miller III, David M., Sestan, Nenad, Taylor, Seth R., Varol, Erdem

[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.

Hung, Wesley L. et al. (2019) International Worm Meeting "A compact circuit of interneurons coordinates two central pattern generators to control forward movement."

McWhirter, Rebecca, Wang, Ying, Hung, Wesley L., Meng, Jun, Chang, Maggie M., Sathaseevan, Anson, Luo, Linjiao, Lu, Yangning, Miller III, David M., Zhen, Mei

[International Worm Meeting, 2019]

In C. elegans, there are two central pattern generators (CPGs) that contribute to forward movement - the head CPG that controls the head swing, through currently unidentified neurons, and the body CPGs, which resides in the B-type motor neurons (Xu et al., 2017). The frequency and amplitude of the head swing and body undulation are tightly coupled to allow smooth, sinusoidal forward movement. We show here that the descending interneurons, AVG and RIF, play a critical role in two aspects of forward movement: forward speed modulation and head-body coordination. While AVG is not essential for locomotion, the loss of AVG results in animals with reduced forward speed and an increased tendency to remain in a pausing/resting state. Conversely, optogenetic activation of AVG alone rapidly increases forward velocity. This effect requires gap junction-mediated activation of RIFL/R, which subsequently activates the premotor interneurons AVBL/R to increase activity of the forward movement-driving B motor neurons. When head swinging is inhibited, body undulation is decreased. Conversely, increased head swinging frequency leads to increased body undulation frequency to potentiate higher forward velocity. This suggests communication between the head and body CPGs. Our preliminary results suggest that AVG may also be required to coordinate the head and body CPGs. Activation of AVG was sufficient to drive body bends even when head swinging was inhibited. Increased head swinging is not able change body undulation when AVG is ablated. We propose that the descending interneuron circuit (AVG-RIF-AVB) permits generation of adaptive forward movement by modulating forward speed and linking the head and body CPGs. Xu, T. et. al. PNAS May 8, 2018 115 (19) E4493-E4502

Hung, Wesley et al. (2015) International Worm Meeting "The C. elegans RID neuron is a neurosecretory cell that modulates locomotion."

Hung, Wesley, Ho, Chi-Yip, Ji, Ni, Lim, Maria A, Samuel, Aravinthan, Zhen, Mei, Chitturi, Jyothsna, Holmyard, Douglas, Calarco, John, Miller, David M., McWhirter, Rebecca, Lu, Yangning, Laskova, Valeriya

[International Worm Meeting, 2015]

* contributed equallyComplex behaviors are modulated by endocrine neurons, which secrete neuropeptides and hormones. Homeostasis of these neuromodulators is critical, and its disruption has been associated with disease. Despite the ancient and significant role that neuromodulators play in complex behaviors across organisms, a comprehensive understanding of neuroendocrine cell function and their regulation is limited. The C. elegans nervous system provides an excellent genetic platform to investigate neural network functions and behavior. However, despite a fully annotated nervous system, a neuron with dedicated neurosecretory properties has not been identified. Using serial transmission electron microscopy, we identified RID as a potential endocrine cell. RID is the only neuron that extends an axon along the full length of the dorsal nerve cord and almost exclusively contains dense core vesicles, which package and secrete neuromodulators. Using a combination of laser ablation, genetics, and imaging techniques, we provide several lines of evidence that the RID neuron is a neuroendocrine cell that modulates motor behaviors. Using transcriptome profiling of a subpopulation of neurons isolated from both wild-type and unc-39 mutant worms, wherein the RID neuron is absent, we found that the RID neuron is enriched with neuropeptides, including flp-14. We confirm that flp-14 is expressed in the RID neuron and that flp-14 mutants possess locomotor defects similar to RID mutants. Our results demonstrate a neuromodulatory role for the RID neuron in regulating locomotion. We propose that the C. elegans RID neuron may be a useful genetic model to probe for conserved molecular mechanisms underlying neuroendocrine cell development and function. .

Rebecca Hall et al. (2006) European Worm Meeting "Identification of pH Regulated Genes in the Nematode Caenorhabditis elegans: New Potential Drug Targets for the Treatment of Pathogenic Nematodes"

Fritz A. Muhlschlegel, Peter Klappa, Rebecca Hall

[European Worm Meeting, 2006]

Rebecca Hall, Peter Klappa and Fritz A. Mhlschlegel. Human pathogenic nematodes infect over 160 million people per year. Brugia malayi and Onchocerca volvulus are the causative agents of elephantiasis and river blindness, respectively. Treatment options are restricted and the most commonly used drug, Ivermection, is stage specific and has significant side effects. Furthermore, treatment failures due to drug resistance have been reported.. Brugia malayi and O. volvulus exhibit an indirect life cycle where an incubation period within an intermediate host vector is essential for development of the infectious stage. During host-vector transition pathogenic nematodes are exposed to extreme environmental changes including variations in pH. We decided to exploit the different environmental pH conditions encountered, in order to identify new therapeutic targets. Using C. elegans as a model we designed a survival assay and were able to show that the nematodes are extremely resistant to environmental pH changes. This result was shown to be independent of the cuticle by the use of collagen cuticle mutants. Microarray experiments identified a set of pH-regulated genes. These genes were up regulated at pH9, an environment encountered by pathogenic nematodes residing in the vector gut. The genes were silenced using RNA mediated interference to establish their requirement for survival. There were no significant phenotypic changes observed after five days of treatment, and exposure of the silenced nematodes to acidic and alkaline environments did not significantly reduce survival. However, in vivo studies are currently being carried out using carbonic anhydrase inhibitors to identify whether the candidate genes have potential for drug targeting.

Varol, Erdem et al. (2021) International Worm Meeting "A computational approach linking neuron-specific gene expression with connectivity"

Miller, David, Reilly, Molly, Taylor, Seth, Paninski, Liam, Poff, Abigail, Varol, Erdem, Hammarlund, Marc, Vidal, Berta, Litwin-Kumar, Ashok, Basavaraju, Manasa, Tavazoie, Saeed, Xu, Chuan, Cros, Cyril, Barrett, Alec, Cook, Steven, Sestan, Nenad, Rafi, Ibnul, Glenwinkel, Lori, Oikonomou, Panos, Weinreb, Alexis, Yemini, Eviatar, Hobert, Oliver, Santpere, Gabriel, Abrams, Alexander, McWhirter, Rebecca

[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., &amp; 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., ... &amp; Miller, D. M. (2020). Molecular topography of an entire nervous system. bioRxiv. 3. Cook, S. J.,... &amp; Emmons, S. W. (2019). Whole-animal connectomes of both Caenorhabditis elegans sexes. Nature, 571(7763), 63-71.