Miller KG et al. (1997) International C. elegans Meeting "RIC AND HIC MUTANTS IDENTIFY POSITIVE AND NEGATIVE REGULATORS OF SYNAPTIC TRANSMISSION"

Miller KG, Emerson MD, Rand JB

[International C. elegans Meeting, 1997]

We are investigating signal transduction pathways that regulate synaptic transmission. We have focused on the pathway(s) that regulate the transmission of acetylcholine (ACh) since ACh is the major excitatory neuromuscular transmitter in C. elegans, and since mutants with altered ACh transmission can be easily identified on the basis of their sensitivity to the acetylcholinesterase inhibitor, aldicarb. Defects that decrease ACh secretion can result in resistance to inhibitors of cholinesterase (Ric). Many Ric genes encode homologs of proteins that function in the basic machinery of neurotransmitter release; however, a subclass of Ric genes (egl-10 and egl-30) encodes molecules that act in G-protein signal transduction pathways, and thus are expected to be positive regulators of ACh transmission (Koelle et al., 1996; Brundage et al., 1996). To identify additional positive regulators, we used chemical and insertional mutagenesis in a series of Ric selections to identify another 8 Ric genes that, in addition to being Ric, share one or more phenotypes with egl-10 or egl-30. To identify the expected negative regulators of ACh transmission, we have developed a screen that identifies mutants that are hypersensitive to inhibitors of cholinesterase (Hic). We have identified 6 Hic genes through this screen and an additional 5 Hic genes by testing mutants in previously identified genes. One Hic gene, goa-1, encodes a molecule that functions in a neuronal G-protein signal transduction pathway (Mendel et al., 1995; Segalat et al., 1995). To begin an analysis of how Ric and Hic gene products interact to regulate ACh transmission, we are making double mutants between various Ric and Hic mutants. Supported by NINDS.

McManus JR et al. (2000) West Coast Worm Meeting "RIC-8 (Synembryn): A novel regulator of G Protein signaling"

McManus JR, Rand JB, Miller KG, Emerson MD

[West Coast Worm Meeting, 2000]

Recent studies describe a network of signaling proteins centered around GOA-1 (Goa) and EGL-30 (Gqa) that regulates neurotransmitter secretion in C. elegans by controlling the production and consumption of diacylglycerol (DAG). We sought other components of the Goa-Gqa signaling network by screening for aldicarb resistant mutants with phenotypes similar to egl-30 (Gqa) mutants. In so doing we identified ric-8, which encodes a novel protein named RIC-8 (synembryn). Through cDNA analysis we show that RIC-8 is conserved in vertebrates. Through immunostaining we show that RIC-8 is concentrated in the cytoplasm of neurons. Exogenous application of phorbol esters or loss of DGK-1 (diacylglycerol kinase) rescues ric-8 mutant phenotypes. A genetic analysis suggests that RIC-8 functions upstream of EGL-30 (Gqa), or in a parallel intersecting pathway. Both ric-8 and goa-1 reduction of function mutants also exhibit partial embryonic lethality. Furthermore, the embryonic lethality of ric-8 mutants is enhanced to 95-100% by a 50% reduction in maternal goa-1 gene dosage. In a separate study we investigated the roles of RIC-8 and GOA-1 in early embryos (pre 8-cell stage) and found that goa-1 and ric-8 mutant embryos exhibit defects in the movements of centrosomes. Comparing the roles of RIC-8 and GOA-1 in the nervous system and the embryo reveals potentially informative differences between the two pathways. First, EGL-30 (Gqa) does not appear to play a role in the embryonic pathway. Second, in the embryonic pathway, reduction of function mutations in goa-1 and ric-8 lead to similar phenotypes that are enhanced in goa-1; ric-8 double mutants, whereas in the adult neuronal pathway the same goa-1 and ric-8 mutants have opposite phenotypes and suppress each other. Based on these findings, we propose that RIC-8 is required, directly or indirectly, for proper activation of G proteins in the early embryo and in the nervous system.

Baritaud, Mathieu et al. (2015) International Worm Meeting "Targeting mitochondria in muscular dystrophies through acting on ROS, mitohormesis and programmed cell death pathways ."

Kretz-Remy, Carole, Pierson, Laura, Baritaud, Mathieu, Walter, Ludivine, Mariol, Marie-Christine, Chazaud, Benedicte, Gieseler, Kathrin, Martin, Edwige

[International Worm Meeting, 2015]

Muscular dystrophies are characterized by muscle weakness and degeneration induced by genetic mutation of muscle structure or signaling components. The development of treatments against dystrophies is challenging, in part due to various primary genetic defects affecting different cellular functions. Nonetheless, many studies observed mitochondrial dysfonctions in muscle pathologies. Moreover, using a worm model of Duchenne Muscular Dystrophy (DMD) our team clearly demonstrated an implication of the mitochondrial key effectors Dynamin Related Protein-1, (DRP-1) and cytochrome c in the early process of muscle degeneration (MD). We are now focusing on Reactive Oxygen Species (ROS) and Programmed Cell Death (PCD) pathways to identify common mitochondrial disorders that contribute to MD in muscular dystrophies. We established different C. elegans models that mimic human muscular dystrophies such as Duchenne and Becker dystrophies, Limb-Girdle, Emery-Dreifuss or congenital muscular dystrophy. We revealed in the C. elegans model for DMD that MD can be reduced by treatments with the mitohormetic compound Paraquat (0,1mM) or the antioxidant compounds NAC and vitamin C. This observation suggested that ROS pathways mediated by mitochondria could be targets to reduce MD. In addition, our preliminary results indicate that these treatments are also efficient against MD in other C. elegans models for human muscular dystrophies. Moreover, using a gain of function mutation in the anti-apoptotic effector ced-9 gene (ortholog of Bcl-2) and RNA interference of key molecular actors of PCD linked to mitochondria: ced-3, ced-4, ced-13 and egl-1, we investigated whether specific mitochondrial PCD pathways are involved in MD. To validate our data obtained in C. elegans, we use human immortalized myoblasts from healthy or DMD patients. Preliminary data indicated that DMD myoblasts proliferate less and have a reduced fusion capacity during myogenic differentiation than myoblasts from healthy patients. Ongoing investigations examine mitochondrial pathways identified in C. elegans by challenging myoblasts for proliferation, cell fusion and oxidative stress sensibility.Our original approach using both worm models and human myoblasts, will allow us to establish whether manipulating mitochondrial pathways, particularly ROS signaling and PCD pathways can reduce progression of MD in muscular dystrophies.

Hongkyun Kim et al. (2002) West Coast Worm Meeting "A Genetic Study of Muscular Dystrophy in C. elegans"

Hongkyun Kim, Steven L McIntire

[West Coast Worm Meeting, 2002]

Muscular dystrophy (MD) is a common genetic disorder characterized by progressive muscle weakness and wasting. Mutations in dystrophin were identified as one cause of this disease in humans. Mutations in the C. elegans homologue of the dystrophin gene, dys-1, cause exaggerated bending of the anterior body during locomotion and muscle degeneration in a sensitized genetic background (1, 2).

Brewer, Jacob et al. (2017) International Worm Meeting "The HSN command neuron co-releases serotonin and NLP-3 neuropeptides to activate egg laying."

Brewer, Jacob, Koelle, Michael

[International Worm Meeting, 2017]

Major depression (MD), one of the most common mental disorders, is highly heritable and is commonly treated with selective serotonin reuptake inhibitors (SSRIs), which increase serotonin signaling in the brain. Yet, genome-wide association studies have failed to definitively implicate genetic defects in serotonin signaling as the cause of MD.1 We are studying the mechanism by which serotonin activates egg laying in C. elegans as a model for understanding the role of serotonin signaling in the activity of neural circuits. The C. elegans egg-laying system is well-suited for studying how serotonin functions in a circuit. The serotonergic HSN motor neurons initiate ~2.5 minute egg-laying active phases that occur every ~20 minutes. The evidence for this is that rhythmic HSN Ca2+ activity measured using a GCaMP reporter always accompanies egg-laying active phases, and optogenetic activation of HSN neurons is sufficient to stimulate activity of the egg-laying circuit.2 Interestingly, while egg laying requires the serotonergic HSNs3 and can be hyperactivated with an SSRI4, mutations knocking out serotonin biosynthesis have only weak effects on egg laying. This puzzling result draws striking parallels with the role of serotonin signaling in MD. We found that the HSNs release a neuropeptide in addition to serotonin to stimulate egg laying. The nlp-3 neuropeptide gene as well as the enzymes necessary for serotonin biosynthesis and release are all expressed in the HSNs. Knocking out either nlp-3 or serotonin biosynthesis caused mild egg-laying defects, but knocking out both caused a strong egg-laying defect that phenocopies that of animals lacking HSNs. Further, we optogenetically stimulated the HSNs and observed that the resulting egg-laying behavior is profoundly dependent on nlp-3, and is completely silenced in animals lacking both NLP-3 neuropeptides and serotonin. We propose that the HSN releases both serotonin and NLP-3 neuropeptides, which act cooperatively in a partially redundant fashion to stimulate egg laying, explaining the absence of strong egg-laying defects in mutants lacking serotonin. Serotonin receptors are found only on the muscles within this circuit, and sensitize them to contract in response to acetylcholine.2 We are currently analyzing the Ca2+ activity in the circuit to determine roles for NLP-3 neuropeptides in circuit activity. Co-release of serotonin and neuropeptides also occurs in the mammalian brain, and by analogy with the worm egg-laying circuit may be an important feature in explaining the absence of a strong genetic link between MD and serotonin. 1Flint and Kindler. Neuron. 2014. 2Collins, et al. eLife. 2016. 3Trent, et al. Genetics. 1983. 4Weinshenker, et al. J. Neurosci. 1995.

Katherine Schouest et al. (2007) International Worm Meeting "Development of a C. elegans model system for genetic and molecular dissection of epigenetic mechanisms underlying trinucleotide expansion disorders."

Charles Spillane, Katherine Schouest, Avril Coghlan

[International Worm Meeting, 2007]

Expansion of nucleotide repeats is now recognized as a cause of at least 20 different developmental and degenerative disorders in humans. Nucleotide-repeat expansion diseases can be classified into 3 main categories. The first disease category consists of loss-of-function recessive mutations due to disruption of gene expression (e.g. Friedrichs ataxia). The second category involves the expansion of nucleotide repeats (e.g. CAG) within exons, which are then translated into poly-amino acid (e.g. polyglutamine) stretches within the corresponding protein (e.g. Huntingtons Disease: HD). The third category involves expansions within non-coding regions of a gene, such as intronic or untranslated regions (e.g. Myotonic Dystrophy: MD). Model organisms are essential for the rapid advancement of genetic research on human diseases. C. elegans has already proven to be a powerful biomedical model in dissecting principles underlying diseases like HD or MD, as well as in revealing potential targets for therapeutic treatment. We are using C. elegans as a model organism to investigate epigenetic phenomena associated with nucleotide repeat disorders and to identify modifiers that regulate the size and rate of the expansions. To identify candidate genes for our research we have screened the C. elegans genome for trinucleotide repeats that have at least 12 trinucleotide repeats and a high level of purity, as these are most likely to undergo expansion or contraction. We have so far identified 20 such repeats located in C. elegans exons and 17 such repeats located within introns. To select one or more variable repeats for further analysis, the polymorphism of these repeats are currently being examined over 48 wild isolates of C. elegans. Once variable repeats have been identified, these alleles will be monitored through multiple generations in control animals and animals depleted of candidate modifiers via RNAi. The project will ultimately facilitate the understanding of the epigenetic phenomena that can cause nucleotide expansion disorders.

Dolinski CM et al. (1997) International C. elegans Meeting "PHYLOGENETIC IMPLICATION OF BUCCAL CAPSULE DEVELOPMENT IN SOME BACTERIAL FEEDING NEMATODES"

Baldwin JG, Dolinski CM, Thomas WK

[International C. elegans Meeting, 1997]

Bacterial feeding nematodes including Zeldia punctata (Rhabditida: Nematoda), and Caenorhabditis elegans differ profoundly in feeding adaptations and specifically in the rhabdions (cuticular thickenings), and associated cells lining in the buccal capsule. We are carrying out a range of tests to determine what rhabdions are evolutionarly homologous between the two species. Reconstruction of the buccal capsule and procorpus (pharynx) with transmission electron microscopy indicates that the lining of the buccal capsule of Z. punctata includes four sets of muscular radial cells ma, mb, mc, md in contrast the same region in C. elegans which has two sets of epithelial radial cells (e1, e3) and two sets of radial muscle cells (m1, m2). In Z. punctata ma is a set of three radial muscle cells each with 2 nuclei. In C. elegans m1 has the identical arrangement. Similarly, in Z. punctata mb is a set of 3 muscle cells each with one nucleus, similar to m1 in C. elegans. These findings could contradict all previous hypotheses of homology, and suggest instead that ma and mb in Z. punctata are homologous respectively with m1 and m2 in C. elegans. Ongoing additional tests of homology include comparative cell lineages and screening mutants to recognize genes involved in buccal capsule expression.

Garriga, G. et al. (2019) International Worm Meeting "Quantitative study of dopaminergic phenotypes via computer-aided video analysis."

Nelms, B., Garriga, G.

[International Worm Meeting, 2019]

Dopamine is an important neurotransmitter that regulates a myriad of brain functions ranging from motor control to memory and learning. Dysregulation of dopamine is associated with multiple disorders including Parkinson's disease (PD), attention deficit hyperactivity disorder (ADHD), addiction, and schizophrenia, among others. In order to better understand dopamine function at the molecular level, we are investigating the roles and regulation of dopaminergic genes using Caenorhabditis elegans (C. elegans) as a model system. We are conducting swimming induced paralysis (SWIP) assays using a computer-aided video analysis system, allowing us to collect more nuanced data to uncover new phenotypes and discover roles for genes involved in dopaminergic signaling. We have selected eight genes to focus on in this study. Each of these eight genes is highly enriched in dopamine neurons, based on RNASeq data from our laboratory, and mutant strains for these genes show accelerated swimming-induced paralysis when compared to the wild-type strain, N2, a phenotype characteristic of hyperdopaminergic signaling. Based on our computer-aided video analysis, we propose to gain more insight into specific rates and patterns of paralysis of these mutant strains in an effort to identify novel genes critical for dopaminergic functioning. Funding support from NSF CREST Grant #HRD1547757, NIH BD2K R25 Grant #1R25MD010396-01, and DOE Title VII Grant MD-HBCU #P382G090004.

KG Miller et al. (1999) International C. elegans Meeting "GOA-1 (Goa) and RIC-8 interact to Regulate Synaptic Transmission in Adults as well as Centrosome and Nuclear Positioning in Early Embryogenesis"

JB Rand, KG Miller

[International C. elegans Meeting, 1999]

Molecular and genetic analyses suggest that the GOA-1 (Go a ) pathway interacts with the EGL-30 (Gq a ) pathway to regulate synaptic transmission. 1 To identify other components of this complex regulatory network, we screened for additional genes that confer phenotypes similar to egl-30 (rf) mutants. Mutants in one such gene, ric-8 , share many phenotypes with egl-30 (rf), including strong aldicarb resistance, reduced body flexion, and strongly reduced rates of locomotion and egg laying. A genetic analysis suggests that, like EGL-30, RIC-8 also interacts with the GOA-1 pathway, although the precise relationship of RIC-8 to the EGL-30 and GOA-1 pathways is not yet clear. In addition to the neuronal phenotypes of ric-8 mutants, both alleles of ric-8 exhibit an incompletely penetrant embryonic lethality (15% for ric-8 (md1909) and 28% for ric-8 (md303) ). Both the neuronal and the embryonic defects disappear in an intragenic revertant of md303 . The embryonic lethality of both alleles is enhanced to 100% when the parental genotype is goa-1/+; ric-8/ric-8 . This suggests that maternally supplied RIC-8 and GOA-1 function together in early embryogenesis. To identify the function(s) disrupted in these mutants, we observed mutant embryos as they progressed from fertilization through the 8-cell stage. ric-8 mutants showed occasional defects in cleavage plane orientation that were secondary to improper centrosome localization and, in addition, showed delayed nuclear positioning. These defects were strongly enhanced in embryos derived from a goa-1/+; ric-8/ric-8 parent. We conclude that GOA-1 and RIC-8 are involved in the production of a signal that regulates the position of centrosomes and nuclei in the large cells of young embryos. We cloned the ric-8 gene and found that it encodes a novel protein that is related to vertebrate EST sequences of unknown function. Immunostaining revealed that the RIC-8 protein is localized throughout the nervous system and in germ cells. 1 Miller, Emerson, and Rand, 1999. C. elegans meeting abstract.

Herrera, R. Antonio et al. (2009) International Worm Meeting "Laser-Capture Microdissection and Microarray Profiling of C. elegans Male Tail Tip Morphogenesis."

Fitch, David H. A., S, Sindhoora, Herrera, R. Antonio

[International Worm Meeting, 2009]

Regulation of C. elegans male tail tip morphogenesis involves the input of several distinct molecular pathways. The heterochronic [1], Wnt signaling [2], Hox patterning [4] and sex determination [3] pathways each contribute to the proper development of the male tail tip syncytium. Mutations in certain genes result in unretracted adult male tail tips. Our lab and others have confirmed the intersection between these molecular pathways by examining the expression of transgenic reporters in various mutant backgrounds [3]. Our current understanding of the genetic network is limited, as many of these interactions are indirect. To identify candidate genes that are involved in tail tip retraction, we are performing a microarray analysis of gene expression in tail tips isolated from synchronized males and hermaphrodites prior to male L4 tail tip morphogenesis. We are using laser-capture microdissection to obtain tail tips, from which total RNA is isolated for hybridization onto Affymetrix C. elegans GeneChips. The tail tip lends itself particularly well to analysis of post-embryonic gene expression in specific somatic cells because the four tail tip cells can be removed by a single slice perpendicular to the anterior-posterior axis. Work is in progress to identify genes that are differentially expressed and/or change in expression profile from late L3 to middle L4. To confirm their role in morphogenesis, we will examine tail tip phenotypes in RNAi knockdowns or mutants of these genes. [1] Del Rio-Albrechtsen et al. 2006, Dev. Biol. 297:74. [2] Zhao et al. 2002, Development 129:1497. [3] Mason et al. 2008, Development 135:2373. [4] see MD Nelson Abstract.