Bai, Xiaofei et al. (2019) International Worm Meeting "Modelling Genetic Diseases of PIEZO Dysfunction in C. elegans."

Bouffard, Jeff, Cram, Erin, Lord, Avery, Golden, Andy, Bai, Xiaofei

[International Worm Meeting, 2019]

PIEZOs are mechanosensitive ion channels; the two PIEZO proteins in humans are involved in a wide range of developmental and physiological processes. Dysfunctional mutations of PIEZOs cause at least six diseases, two of which are Dehydrated Hereditary Stomatocytosis (DHS) and Generalized Lymphatic Dysplasia (GLD). However, the cellular and molecular mechanisms of PIEZOs in these diseases are less understood. To address this, we investigated the functional roles of pezo-1, the sole PIEZO ortholog in C. elegans, using CRISPR/Cas9 and other genetic tools. GFP::PEZO-1 widely expresses from embryos to adults in C. elegans. Notably, it strongly expresses at several valvular tissues, including pharynx-intestine and spermatheca-uterine valves in C. elegans. It is also widely expressed in the somatic gonad and sperm. Two large deletions and multiple patient-specific loss-of-function and gain-of-function mutants cause severe reproductive deficiency. In vivo observations show that nascent fertilized oocytes underwent a variety of transit defects in and out of the spermatheca. Post ovulation oocytes were frequently damaged during spermathecal contraction. Due to PIEZO channels' permeability to Ca2+ and the importance of calcium signaling in regulating spermatheca contractility, we are imaging the calcium indicator GCaMP in our pezo-1 mutants. Variation of calcium release was observed in our pezo-1 mutants compared with wild type worms. Therefore, our results suggest that the mechanosensitive channel PEZO-1 coordinates cellular contractility to promote proper ovulation in C. elegans. Finally, we will attempt genetic and chemical suppressor screens to identify putative suppressors in pezo-1 mutants. These screens will use FDA-approval drugs to suppress the mutant phenotypes, hopefully providing insightful ideas for future clinical therapy.

Bai, Xiaofei et al. (2021) International Worm Meeting "Deciphering the functional roles of PIEZO mechanosensors in reproduction"

Brugman, Katherine, Golden, Andy, Bouffard, Jeff, Lord, Avery, Bai, Xiaofei, Cram, Erin, Sternberg, Paul

[International Worm Meeting, 2021]

There are > 6000 rare (or orphan) monogenic diseases identified in humans; many of these diseases are caused by the dysfunction of channel proteins, known as channelopathies. A good example is the recently identified mechanosensitive channels PIEZO1 and PIEZO2, of which ~100 missense mutations are associated with at least 26 dysplasias/diseases in the muscular, lymphatic, connective, and cardiovascular tissues. PIEZO1/2 are excitatory mechanosensitive proteins; they are non-selective ion channels that exhibit a preference for calcium in response to mechanical stimuli. Dysfunction of PIEZOs cause a variety of genetic diseases, including the dysplasia in cardiovascular, respiratory, and connective tissues. However, the cellular and molecular mechanisms of PIEZOs in these diseases are less understood. To further understand the function of these proteins, we investigated the roles of pezo-1, the sole PIEZO ortholog in C. elegans. pezo-1 is expressed throughout development in C. elegans, with strong expression in reproductive tissues, including spermatheca, oocyte, sperm, and somatic sheath cells. A number of deletion alleles as well as a putative gain-of-function mutant caused severe defects in reproduction. A reduced brood size was observed in the strains depleted of PEZO-1. In vivo observations show that oocytes undergo a variety of transit defects as they enter and exit the spermatheca during ovulation. Post ovulation oocytes were frequently damaged during spermathecal contraction. Given that PIEZO is an ion channel and may regulate spermathecal contractility through Ca2+ signaling pathways, we tested the genetic interactions between pezo-1 mutants and several cytosolic Ca2+ regulators with RNA interference (RNAi). Indeed, the phenotypes of pezo-1 mutants are enhanced upon depletion of known cytosolic Ca2+ regulators. We also observed that loss of PEZO-1 revealed an inability of self-sperm to properly navigate back to the spermatheca after being pushed out of the spermatheca during ovulation. Mating with males rescued these reproductive deficiencies in our pezo-1 mutants. Reduced brood sizes were also observed in a number of auxin-inducible tissue-specific degradation strains, suggesting PEZO-1 may act in different reproductive tissues to coordinate reproduction. Using CRISPR/Cas9, we generated the patient-specific PIEZO2 allele (p.R2718P) in C. elegans, named pezo-1(R2405P). Homozygous animals carrying the pezo-1(R2405P) mutation displayed reproductive defects similar to the pezo-1ko mutants, including reduced ovulation rates, crushed oocytes in the uterus, and reduced brood sizes. Overall, these observations support the idea that C. elegans is an appropriate model system to study PIEZO diseases. An undergoing EMS-mediated suppressor screen with this pezo-1 patient-specific allele should help identify other genetic interactors.

G Aspoeck et al. (1999) International C. elegans Meeting "Does the ortholog of the brain development genes ems/EMX have a function in C. elegans?"

TR Buerglin, G Aspoeck

[International C. elegans Meeting, 1999]

The Drosophila empty spiracles (ems) and the vertebrate Emx1 and Emx2 genes define brain regions in a way that brain parts expressing these genes are largely missing in loss-of function mutants. Additionally, ems is involved in tracheal development, and Emx2 knockout mice completely lack a urogenital system. Burglin et al. (1989) first identified ceh-2 in a screen for C. elegans homeobox genes. With the ems/Emx genes ceh-2 shares a hexapeptide, 80% identity in the homeodomain and flanking regions as well as an intron at a conserved position. However, the function of ceh-2 does not seem to be comparable to ems/Emx: Anti-peptide antibody staining and expression of gfp/lacZ reporter constructs are restricted to a subset of cells in the pharynx, muscle m2, epithelial e2, and the neurons I3, M3 l/r, and NSM l/r. In an EMS mutagenesis screen we isolated ceh-2(ch4), a 2.5 kb deletion that covers the hexapeptide and homeodomain. The mutant is perfectly viable and fertile and shows no obvious phenotype. What might ceh-2 do? Among the ceh-2 expressing cells, M3 and NSM have been described in more detail. M3 l/r are inhibitory glutamatergic neurons that controls pharynx muscle relaxation. Laser ablation of M3 causes the pharynx muscle to relax more slowly after contraction, a pump cycle is about 10% longer and the electropharyngeogram lacks several inhibitory post-synaptic potentials (Avery, 1993; Raizen and Avery, 1994). Although ceh-2(ch4) mutants do not look starved as M3 ablated animals do, it may be useful to test for M3 activity in ceh-2(ch4). NSM l/r contain serotonine and stimulate pumping; we will test ceh-2(ch4) for corresponding defects. In addition we will express ceh-2 in Drosophila ems mutants to test whether ceh-2 can rescue some of the fly phenotypes. References: Avery (1993) J. Exp. Biol. 175:283-297 (and many WBG abstracts) Burglin et al. (1989) Nature 341:239-243 Raizen and Avery (1994) Neuron 12:483-495

Dent JA et al. (1995) International C. elegans Meeting "The Pharyngeal Motor Neuron M3 is an Inhibitory Glutamatergic Neuron"

Davis MW, Avery L, Dent JA

[International C. elegans Meeting, 1995]

The M3s are a pair of pharyngeal motor neurons that synapse onto corpus muscle. M3 activity results in inhibitory post-synaptic potentials (IPSPs) in the muscle that can be measured by electropharyngeogram (EPG). Ablating M3 with a laser eliminates these IPSPs and results in a longer period of muscle depolarization and contraction during a pump (Avery J. Exp. Biol. 175:283, Raizen & Avery Neuron 12:483). To identify the M3 neurotransmitter, we developed an assay for the acute effect of neuro- transmitters on pharyngeal muscle; the pharynx is dissected out and a pulse of neurotransmitter is iontophoresed onto the depolarized pharyngeal muscle. Glutamate, but not aspartate, mimics the action of the M3 neurons by shortening the duration of the muscle depolarization. This effect occurs even when the M3 neurons are laser ablated and depends on the external chloride concentration. Two mutants lack M3-derived IPSPs: we identified eat-4 in a screen for starved worms (Avery Genetics 133:897) and we identified avr-15(ad1051) in a screen for worms that have abnormal EPGs. We tested both mutants for glutamate sensitivity: dissected pharynxes from eat-4(ad572) worms were still sensitive to iontophoretically applied glutamate but pharynxes from avr-15(ad1051) worms were not. Because iontophoretically applied glutamate mimics the effects of the M3 neurons and because pharyngeal muscle that is insensitive to glutamate also lacks M3 neurotransmission, we believe the M3s are fast inhibitory glutamatergic neurons. avr-15 appears to be necessary post-synaptically for the function of a glutamate-gated chloride channel whereas eat-4 appears to be necessary pre-synaptically for M3 activity.

Rankin CH et al. (1997) International C. elegans Meeting "THE ROLE OF GLUTAMATE TRANSMISSION IN HABITUATION OF THE TAP WITHDRAWAL RESPONSE"

Wicks SR, Rankin CH

[International C. elegans Meeting, 1997]

We have been investigating the effects of mutations in 3 genes on habituation to tap. These mutations, suggested to us by Leon Avery, are eat-4, avr-14 and avr-15; all have been hypothesized to be involved in inhibitory glutamate transmission . In earlier work (Wicks and Rankin, 1997) we showed that the most likely site of habituation was the chemical synapses from the touch cells onto the interneurons, and that these synapses were most likely to be inhibitory (Wicks and Rankin, 1995). The eat-4 gene is thought to encode a transporter required for glutamate neurotransmission (Lee and Avery, pers. comm.). Our results suggest that, although the initial levels of responding are normal in eat-4 animals, habituation of the tap withdrawal reflex is broadly affected suggesting that glutamate transmission may be involved in habituation of the touch receptors. Habituation of eat-4 animals is very rapid compared to wild-type worms. Recovery from habituation in eat-4 animals is also delayed. The avr-15 gene is thought to encode the a glutamate-gated chloride channel subunit. Avery (pers. comm.) has hypothesized that the eat-4 is involved in glutamate transport in the pre-synaptic neurons and that avr-15 affects the post-synaptic inhibitory glutamate receptor. Avr-15 worms respond normally to tap, but show a slightly slower rate of habituation than for wild-type worms. We have also begun testing avr-14 worms (hypothesized to interact with avr-15; C. Johnson, pers. comm.) as well as the avr-14;avr-15 double strain. With the avr-14;avr-15 double we see a slower rate of habituation and a more rapid rate of recovery than for wild-type worms.

Brundage LA et al. (1998) West Coast Worm Meeting "EAT-11, A POSSIBLE NEGATIVE REGULATOR OF EGL-30 (GqA) SIGNALLING"

Sternberg PW, Brundage LA, Simon MI

[West Coast Worm Meeting, 1998]

egl-30 encodes a G protein a subunit which is more than 80% identical to two members of the mammalian Gqa family[1]. We are interested in genes which act in the egl-30 pathway, as they may encode new regulators of G protein signalling. eat-11 is one such gene. eat-11 animals arrest at hatching in the presence of low levels of the cholinergic agonist arecoline[2] and mutations in egl-30 strongly suppress this arecoline effect[1]. We have found that eat-11 animals exhibit behaviors which are opposite to egl-30(r.f). animals in several respects. egl-30(r.f.) animals move slowly and with reduced flexations, and lay few eggs in the presence of serotonin. By contrast, eat-11 animals move more quickly than wild type animals and with exaggerated flexations. In addition, eat-11 animals respond dramatically more strongly than N2 to serotonin in an egg laying assay. We hope to clarify the relationship between egl-30 and eat-11 by determining the molecular identity of the EAT-11 protein. [1] Brundage, L. ,Avery, L., Katz, A., Kim, U-J., Mendel, J.E., Sternberg, P. W., and Simon, M.I. (1996) Mutations in a C. elegans Gqa gene disrupt movement, egg laying, and viability. Neuron 16: 999-1009. [2] Avery, L. (1993) The genetics of feeding in Caenorhabditis elegans. Genetics 133: 897-917.

Conroy, Brian et al. (2013) International Worm Meeting "The neuronal basis of food choice behavior in Caenorhabditis elegans."

Morabe, Maria, Chambers, Melissa, Conroy, Brian, Macfarlane, Rachel, Haynes, Lillian, Glater, Elizabeth

[International Worm Meeting, 2013]

Caenorhabditis elegans uses chemosensation to distinguish among various species of bacteria, their major food source (Ha et al., 2010; Shtonda and Avery, 2006). Although the neurons required for the detection of specific food-odors have been well-defined (Bargmann, 2006), less is known about the sensory circuits underlying the discrimination among the mixtures of odors released by bacteria. We plan to examine the neural machinery underlying bacterial preference among a diverse set of bacterial species. Does bacterial choice use one common neuronal mechanism or a diversity of mechanisms depending on the bacteria? Do some bacterial choices involve a single sensory neuron and others involve multiple sensory neurons? To address these questions, we are testing the food preferences of C. elegans for bacteria found in their natural habitats (kindly provided by Marie-Anne Felix, Institut Jacques Monod, Paris, France). We have found that C. elegans strongly prefers the odors of Providencia sp., Alcaligenes sp., and Flavobacteria sp., to Escherichia coli HB101, a commonly used food source for C. elegans. We have identified that the olfactory neuron AWC is necessary for this preference. We intend to test whether other amphid sensory neurons are also necessary for bacterial preference. In the future we will extend our analysis to other bacterial species to determine the diversity of the underlying neuronal mechanisms. Bargmann, C.I. (2006). http://www.wormbook.org. Ha, H.I., Hendricks, M., Shen, Y., Gabel, C.V., Fang-Yen, C., Qin, Y., Colon-Ramos, D., Shen, K., Samuel, A.D., and Zhang, Y. (2010). Neuron 68, 1173-1186. Shtonda, B.B., and Avery, L. (2006). J Exp Biol 209, 89-102.

Kim WJ et al. (1991) International C. elegans Meeting "IVERMECTIN RESISTANCE IN C. ELEGANS"

Kim WJ, Johnson CD

[International C. elegans Meeting, 1991]

Ivermectin is a potent anthelmintic that prevents growth of C. elegans by blocking pumping of the pharynx (first noted by M. Chalfie; Avery and Horvitz, (1990) J. Exp. Zool. 253; 263-270; L. Avery pers. comm.). Animals resistant to ivermectin are easily selected by growing eggs on ivermectin containing media. Our experiments are designed to evaluate the incidence of spontaneous and induced resistance to various concentrations of ivermectin and to analyze the phenotypes of ivermectin resistant mutants. The results have implications for ivermectin-use programs and may provide tools for the isolation and identification of the ivermectin receptor. Recent experiments have focussed in two areas: detailed examination of the effects of ivermectin near the threshold for growth inhibition and genetic analysis of high-level resistant mutants. Near the threshold, ca. 2.0 ng/ml, the response of individual larvae appears to be 'all or none': Some individuals arrest growth immediately upon hatching; others grow, at normal or near normal rates, to healthy, fertile, adults. Slow growing or sick animals are less frequently observed. Further, the progeny of many, apparently resistant, animals respond to ivermectin exactly as wild type animals. The incidence of this 'non-heritable' resistance, to 5 ng/ml ivermectin, is ca. 1 in 500 eggs, 20X the incidence of heritable resistance. The incidence of heritable, but not of non-heritable, resistance is increased in strains with active transposons (TR403, RW7097) or after EMS treatment. Resistance to 100 ng/ml ivermectin is very rare -- less than 1 in 10( 9) (spontaneous) and ca. one in 30 x 10(6) genomes (after EMS). Most ( perhaps all) high level resistance requires multiple mutations. To date, we have identified 5 genes, including two X-linked uncs, which contribute mutations conferring this phenotype.

Aamna Kaul et al. (2003) International Worm Meeting "Hyperactivation of glutamate-gated chloride channels with ivermectin results in necrosis."

Joseph Dent, Aamna Kaul

[International Worm Meeting, 2003]

Ivermectin is a widely used antiparasitic drug. It kills worms by activating glutamate-gated chloride channels (GluCls), which belong to the family of ligand-gated anion channels that includes the GABA and glutamate receptors (Cully et al., 1994; Dent et al., 2000). The chloride permeability that ivermectin induces in excitable cells tends to prevent excitation. For example, ivermectin targets a GluCl expressed in the pharyngeal muscle to inhibit muscle contraction and prevent eating (Dent et al., 1997). The worms linger for several days in the presence of ivermectin before they starve to death. However, we have found that the lethal effects of ivermectin on C. elegans become irreversible after only a few hours of exposure. When L1 worms were exposed to 20ng/ml for 5 hours and then washed, they gradually developed large vacuoles in their pharyngeal muscle over the next several days. A mutant strain that lacks ivermectin receptors shows little or no necrosis when treated. Ivermectin is hydrophobic and it irreversibly opens GluCls expressed in Xenopus oocytes. So it is possible that ivermectin persists in membranes and continues to activate GluCls. Furthermore, it has been shown that hyperactive cation channels can induce excitotoxic necrosis (Driscoll and Chalfie, 1991). Why, though, would an inhibitory channel have a similar effect when hyperactivated? We are trying to address this question by looking at whether mutations known to inhibit excitotoxicity also inhibit the necrotic effects of ivermectin. Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LHT, Schaeffer JM, Arena JP. Nature 371: 707-711 1994 Dent JA, Smith MM, Vassilatis DK, Avery L. PNAS USA 97: 2674-2679 2000 Dent JA, Davis MW, Avery L. EMBO Journal 16: 5867-5879 1997 Driscoll, M and Chalfie, M. Nature 349: 588-593 1991

Tang XX et al. (1997) International C. elegans Meeting "INTESTINAL BRUSH BORDER TWITCHIN"

Benian GM, Fleming JT, Heierhorst J, Tang XX

[International C. elegans Meeting, 1997]

Previously we noted that a new twitchin is encoded by cosmids F17A9 and F12F3 on chromosome V. Sequence obtained from the Sequencing Project was analyzed by GeneMark and our sequencing of RT-PCR products and cDNA clones. We can account for coding sequence which specifies a 7,030 amino acid polypeptide distributed over a 36,983 bp gene interrupted by 29 introns. The tenth intron (9,497 bp) contains the coding sequence for the mutationally-defined exp-2 potassium channel (L. Avery) on the opposite strand. New twitchin consists of 33 Ig, 7 FnIII domains, and a protein kinase domain. It also has a 1426 residue region containing 8 copies of a ~30 residue motif. Bacterially expressed kinase catalytic core has protein kinase activity towards peptides derived from myosin light chains, with a Vmax similar to the Vmax of constitutively active twitchin kinase. The 60 amino acids lying just C-terminal to the kinase domain partially inhibit kinase activity. Adding 15 residues to the beginning of these 60 residues, by alternative splicing, relieves much of this inhibition. A polyclonal antiserum (EU48) reacts to a twitchin-sized polypeptide on western blots and localizes to the intestinal brush border by immunofluorescent staining. In fact, EU48 co-localizes with monoclonal MH33 (R. Francis) which was shown previously to be restricted to the terminal web. In vertebrates, the terminal web is a region where the microvillar actin bundles are anchored in a complex network of filaments containing myosin II, spectrins, tropomyosin, microtubules, intermediate filaments, and a molecule similar to titin (a twitchin homolog). Several lethals with death at early larval stages have been mapped by Avery et al. to either side of exp-2 (-0.32 mu), the closest being let-402 (-0.34 mu). After determining the 5' end of the gene, we will try to rescue these lethals with new twitchin.