Alexander Bounoutas et al. (2007) International Worm Meeting "mec-15 encodes an F-box protein with WD repeats required for proper touch receptor neuron mechanosensation, morphology, and synapse development."

Michael L. Nonet, Alexander Bounoutas, Kun Zheng, Martin Chalfie

[International Worm Meeting, 2007]

The mec-15 gene was originally identified in a screen for touch insensitive mutants. mec-15 mutations cause partial touch insensitivity that is temperature sensitive. The sam-3 mutation was identified in an independent screen for animals with mislocalized presynaptic markers in the touch receptor neurons; this phenotype is also temperature sensitive. mec-15 and sam-3 are the same gene, since mec-15 and sam-3 alleles fail to complement each other for partial touch insensitivity and presynaptic marker mislocalization. All alleles also affect touch receptor neuron morphology, causing an enlargement of the cell body. We used Snip-SNP mapping and gene sequencing to identify T01E8.4 as mec-15/sam-3. T01E8.4 encodes a 406 a.a. protein containing an F-box domain and WD repeats. Interestingly, F-box proteins, which act as protein substrate adaptors in ubiquitin ligase complexes, have been implicated in C. elegans ventral cord synapse formation (Liao et al. Nature 430: 345-350, 2004). All mec-15/sam-3 alleles save one are nonsense mutations in the T01E8.4 coding sequence; the remaining allele has two missense mutations. The mutant alleles in trans to a deletion of the region do not produce an enhanced phenotype, suggesting that the alleles are functional nulls. Transforming mec-15 mutants with the T01E8.4 gene rescues their mechanosensitive abnormal phenotype. The protein-GFP fusion is expressed in several neurons including the touch receptor neurons, where its expression is cytoplasmic, absent in adults, and dependent on the MEC-3 transcription factor. We are examining gene interactions to identify proteins that are needed for mec-15 action. Since a p38 MAP kinase pathway has been implicated in ventral cord synapse development (Nakata et al. Cell 120: 407-420, 2005), we examined mec-15 phenotypes in a pmk-3 background; all the phenotypes were enhanced. In addition, loss of one copy of the mec-7 <font face=symbol>b</font>-tubulin gene suppresses mec-15 phenotypes, suggesting an interaction between MEC-15 and the touch receptor neurons'' specialized microtubules.

Rui Wang et al. (2005) International Worm Meeting "Identifying genes required for the function of glutamatergic synapses"

Rui Wang, Andres Maricq, Yi Zheng, Penelope Brockie

[International Worm Meeting, 2005]

Glutamatergic neurotransmission accounts for the vast majority of excitatory synaptic signaling in the vertebrate central nervous system. An important problem in neurobiology is to understand how glutamatergic synapses are formed and regulated. Recently, molecules required for activity of ionotropic glutamate receptors (iGluRs) have been identified, shedding more light on the mechanisms underlying the regulation of iGluRs. However, we still have much to learn regarding how iGluRs are synthesized, assembled, localized to the surface of appropriate synapses, and how receptor function is regulated once at the synapse. To address these questions, we have undertaken a genetic screen for modifiers of a dominantly active variant of the GLR-1 iGluR subunit in C. elegans. GLR-1 is expressed in many neurons, including the command interneurons that control locomotion. Introducing an alanine (A) to threonine (T) mutation in a highly conserved site of GLR-1 causes transgenic lurcher worms that express the GLR-1(A/T) variant to rapidly switch between forward and backward movement (Zheng et al., 1999). This dramatic phenotype provided a tool used in a suppressor screen to identify genes required for GLR-1 function. Thus, the sol-1 (suppressor of lurcher) gene was isolated (Zheng et al., 2004). In an attempt to enhance the suppressor screen, we have engineered a second mutation, glutamine (Q) to tyrosine (Y), in the GLR-1(A/T) variant. The GLR-1(Q/Y) mutation inhibits receptor desensitization (Brockie et al., 2001). Transgenic worms that express GLR-1(Q/Y; A/T) have a super-lurcher phenotype, switching between forward and backward movement at a higher frequency than transgenic GLR-1(A/T) worms. We have also tagged GLR-1(Q/Y; A/T) with GFP to facilitate the characterization of genes identified in the suppressor screen. We have screened approximately 15,000 mutagenized GLR-1 (A/T; Q/Y) haploid genomes and isolated 5 candidate suppressors of the super-lurcher locomotion phenotype. We are currently mapping and cloning these genes. Brockie et al. (2001) Neuron 31: 617-30 Zheng et al. (1999) Neuron 24: 347-61 Zheng et al. (2004) Nature 427: 451-7

Rui Wang et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Identifying Genes Required for Functional Glutamatergic Synapses in C. elegans"

Andres Maricq, Rui Wang

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Glutamate is the major excitatory neurotransmitter in the vertebrate CNS and has been implicated in many processes, including synaptic plasticity and neurodegenerative diseases. Therefore, it is important to understand how glutamatergic synapses are formed and regulated. To address this, we have undertaken genetic screens to identify components required for the synthesis, assembly, localization and function of GLR-1, an ionotropic glutamate receptor (iGluR) subunit in C. elegans. GLR-1 is expressed in the command interneurons that control locomotion. Transgenic worms that express the dominant GLR-1(A687T) variant, named "lurchers", rapidly switch between forward and backward movement (Zheng et al., 1999). This dramatic phenotype was used in a genetic suppressor screen that isolated sol-1 (suppressor of lurcher) - a CUB-domain transmembrane protein that is required for GLR-1 function (Zheng et al., 2004). In order to identify additional genes required to build a glutamatergic synapse, we have continued the screen. We screened approximately 4,000 mutagenized haploid genomes and isolated 6 candidate suppressors of the lurcher locomotion phenotype. Three of these fall into a single complementation group that we have mapped to CHR X. We are currently attempting to clone this gene as well as other suppressor mutations.

Penelope J Brockie et al. (2004) West Coast Worm Meeting "Identifying Genes Required for Glutamatergic Neurotransmission"

Andres V Maricq, Jann Gardner, Penelope J Brockie

[West Coast Worm Meeting, 2004]

A challenging problem in the field of neuroscience is to understand how a functional synapse is formed. This complex process involves many steps, including the differentiation of numerous cell types, the establishment of synaptic connections, and the localization of molecules, such as ligand-gated ion channels, to the appropriate synapses. It is critical that these processes be carried out with great precision in order for the nervous system to function properly. We have undertaken a genetic approach to identify molecules that are required to build a glutamatergic synapse. Recently, we described a novel transmembrane protein, SOL-1, that is required for glutamatergic neurotransmission in C. elegans (Zheng, et al., 2004). The sol-1 gene was isolated in a genetic screen for suppressors of GLR-1(A/T) - a dominantly active variant of the GLR-1 ionotropic glutamate receptor (iGluR) subunit. GLR-1(A/T) carries an alanine to threonine mutation that results in a partially ligand-independent ion channel (Zheng et al., 1999). Transgenic worms that express GLR-1(A/T) have severe locomotion defects characterized by rapid switching between forward and backward movement. A null mutation in sol-1 suppresses the hyper-reversal behavior such that mutant worms reverse at a similar frequency to wild-type. Electrophysiological analysis revealed that SOL-1 is required for GLR-1 dependent glutamate-gated currents in the AVA interneurons. The AVA neurons are one of 5 pairs of command interneurons that express GLR-1. Interestingly, the sol-1 mutation does not effect GLR-1 expression, synaptic localization or membrane insertion, and may have a role in receptor gating. By screening for additional suppressors of the GLR-1(A/T) hyper-reversal phenotype, we hope to identify novel genes that are necessary for the function of iGluRs. We expect to find genes required for GLR-1 expression and thus neuronal differentiation. We also anticipate the identification of genes that function to traffic GLR-1 from the cell body to the appropriate post-synaptic sites. C. elegans expresses both non-NMDA and NMDA iGluRs - the two major receptor subtypes. GLR-1 is a member of the non-NMDA class, whereas NMR-1 and NMR-2 belong to the NMDA class. We have undertaken a similar approach to that described above to identify genes required for NMDA receptor function. By introducing various mutations in the NMR-1 and NMR-2 subunits, we hope to generate transgenic worms with locomotion defects. Genes that suppress these defects when mutated are predicted to encode molecules involved in the regulation of this important class of ligand-gated ion channels. Zheng, et al., (1999). Neuron 24(2), 347-361. Zheng, et al., (2004). Nature 427(6973), 451-457.

Jann W Gardner et al. (2005) International Worm Meeting "Identification of Novel Molecules Required for Glutamatergic Synaptic Signaling Mediated by Kainate Receptors"

Jann W Gardner, Craig S Walker, Andres Villu Maricq

[International Worm Meeting, 2005]

The formation of a functional glutamatergic synapse is a complex process involving many steps, including cell differentiation, formation of synaptic connections, and the localization of specific proteins to the appropriate synapses. C. elegans expresses the two major classes of ionotropic glutamate receptors (iGluRs), NMDA and non-NMDA. Non-NMDA receptors can be further classified into AMPA, kainate and delta subtypes. The GLR-1 subunit belongs to the AMPA class and is expressed in many neurons. Conversely, the kainate receptor subunits GLR-3 and GLR-6 are exclusively expressed in the RIA interneuron pair. Much less is currently known about the regulation of kainate receptors; however, available evidence suggests that they are regulated differently than AMPA receptors. Previously, we described a novel CUB-domain protein, SOL-1, that is required for function of GLR-1 receptors (Zheng et al., 2004). sol-1 was isolated using a genetic screen for suppressors of a dominantly active GLR-1 variant, which has an alanine to threonine mutation in a highly conserved region of iGluRs. In an effort to identify genes important for kainate receptor assembly and function, we have undertaken a similar gain-of-function approach.The alanine to threonine mutation was made at the conserved site in GLR-3 and GLR-6 subunits. Transgenic worms expressing GLR-6(A/T) show a locomotion defect characterized by slow, loopy movements. We have also expressed functional heteromeric GLR-3 and GLR-6 receptors in Xenopus oocytes. Interestingly, these receptors function independently of SOL-1, suggesting that AMPA and kainate receptors are regulated differently. Currents recorded from oocytes that express GLR-6(A/T)/GLR-3 or GLR-6/GLR-3(A/T) have dramatically slower kinetics of desensitization compared to wild-type.By screening for suppressors of the movement defects in transgenic worms that express GLR-6 (A/T) and GLR-3(A/T), we hope to identify genes necessary for kainate receptor expression as well as genes that function in the assembly, localization and membrane insertion of these receptors. Zheng et al., (2004) Nature 427: 451-7

Cheon, Y. et al. (2019) International Worm Meeting "GDE-4 mediates pheromone avoidance behavior of C. elegans."

Kim, K, Cheon, Y., Park, Y.

[International Worm Meeting, 2019]

The glycerophosphodiesterases (GDEs) hydrolyze water-soluble glycerohposphodiesters and GPI anchored proteins, of which hydrolyzed substrates regulate signal transduction, osmoprotection, motor-neuronal differentiation, cytoskeletal organization and osteoblast differentiation (Corda et al., 2014). However, functions of GDEs in nervous systems are not fully understood. We first performed BLAST searches against the C. elegans genome with sequences of mammalian GDE domain and identified five orthologous genes (Zheng et al., 2002), which we named as gde-1-5. We then examined expression patterns of these genes by generating transgenic animals expressing gfp reporter transgenes under the control of their own promoter. We found that gde-4 was specifically expressed in the ADL chemosensory neurons. We and other previously showed shown that the ADL neurons detect ascaroside pheromone ascr#3 (C9) (Jang et al., 2012). To investigate role of GDE-4 in ADL, we generated gde-4 null mutant animals and found that gde-4 mutants fail to exhibit ascr#3 avoidance behavior. Currently, we are performing rescue experiments by expressing gde-4 cDNA in ADL, and calcium imaging of ADL in gde-4 mutant animals.

D Messerschmidt et al. (2004) European Worm Meeting "ARE MIG-5 AND POP-1 INVOLVED IN PRISTIONCHUS PACIFICUS VULVA DEVELOPMENT?"

R Sommer, D Messerschmidt

[European Worm Meeting, 2004]

To study the evolution of developmental processes we compare vulva development between the nematodes Caenorhabditis elegans (Rhabditidae) and Pristionchus pacificus (Diplogastridae), which shared a last common ancestor around 300 million years ago. In C. elegans, the EGF/RAS/MAPK-pathway is involved in vulva induction. In contrast, it is not clear whether EGF- or other signalling pathways are required to induce vulva development in P. pacificus. In genetic screens for vulva defective phenotypes, P. pacificus mutations in the lin-17/Frz-gene were isolated (Zheng, Roeseler and Sommer, see contribution to this meeting). This result suggests that Wnt-signalling is one of the signalling pathways necessary for vulva development in P. pacificus.Using degenerate primers, we cloned several possible downstream interactors of the WNT-signalling pathway. For further investigation of these genes, we are taking two different approaches. First, we started to analyze the function of mig-5/Dsh, apr-1/APC and pop-1/TCF by Morpholino knockdown experiments. We observed an increase in frequency of bivulva phenotype after injection of mig-5 Morpholino in a sensitized background. Second, we are investigating proteins that might interact with POP-1 using yeast two-hybrid screens. With this approach we expect to find more components involved in Wnt signal transduction in P. pacificus. These experiments might provide insight into the molecular evolution of vulva induction resulting in different patterns such as those found in P. pacificus and C. elegans.

Kim Reese et al. (2001) International C. elegans Meeting "A novel protein required for degradation of CCCH finger proteins in somatic lineages."

Geraldine Seydoux, Kim Reese

[International C. elegans Meeting, 2001]

The first cleavages of the embryo are asymmetric and give rise to 5 somatic lineages and the embryonic germ lineage. Several maternal proteins are asymmetrically segregated during these cleavages: in particular three proteins, PIE-1, POS-1 and MEX-1, segregate preferentially with the germ lineage and are excluded from somatic lineages. These proteins have in common a pair of CCCH zinc fingers (ZF1 and ZF2). We have found that, when fused to GFP, ZF1s from PIE-1, MEX-1 and POS-1 cause GFP to be degraded specifically in somatic blastomeres. Consistent with a role in protein degradation, mutations in PIE-1 ZF1 cause abnormal stabilization of PIE-1 in somatic blastomeres. These observations suggest that the ZF1 in PIE-1, MEX-1 and POS-1 targets these proteins for degradation specifically in somatic blastomeres. To identify factors that function in this process, we performed a yeast-two hybrid screen for proteins that interact with PIE-1 ZF1 and identified a novel protein, F59B2.6 (thanks to Zheng Zhou and Bob Horvitz for their excellent library). F59B2.6 does not appear to have any recognizable motifs. GST pull down experiments have confirmed that F59B2.6 interacts directly and specifically with ZF1 and does not interact with ZF2. RNA-mediated interference of F59B2.6 causes abnormal accumulation of PIE-1,MEX-1 and POS-1 in somatic blastomeres, and results in embryonic lethality. F59B2.6(RNAi) embryos show no defects in PAR-2, PAR-6 and GLP-1 localization, indicating that this gene is not generally required for embryonic polarity. These observations suggest that F59B2.6 recognizes ZF1-containing proteins and targets them for degradation in somatic blastomeres. Although CCCH fingers in other proteins (e.g. mammalian TIS11) have been implicated in binding to RNA, our findings raise the possibility that CCCH fingers can also function as determinants of protein stability.

Maricq AV et al. (2000) West Coast Worm Meeting "Analysis of GLR-7, GLR-5, Ionotropic Glutamate Receptor Subunits"

Walker CS, Maricq AV, Brockie PJ, Madsen DM

[West Coast Worm Meeting, 2000]

Of the ten putative ionotropic glutamate receptor subunits identified in the worm, two non-NMDA subunits, glr-5 and glr-7, show a very restricted expression pattern. When GLR-5 and GLR-7 are fused to GFP they show expression in only a single cell, the interneuron RIA. RIA receives synaptic input from the neurons AIY and AIZ. Previous studies have defined this group of cells to form part of the circuit that controls thermotaxis. To determine the possible roles of glr-5 and glr-7 in thermotaxis, we have generated deletion mutations in both genes. These deletions remove a portion of the extracellular domain and the first three transmembrane domains. In each case the mutations result in a functional null. Analysis of glr-5(ak57) deletion mutants using thermotaxis assays is underway. glr-5(ak57) worms also appear to have a diminished brood size, which is more severe at elevated temperatures. In order to examine the role of RIA in processing thermal information we have generated transgenic strains which express an activated form of the GLR-1 glutamate receptor subunit, GLR-1 (A/T), under control of the glr-5 promoter. GLR-1 (A/T) causes constant depolarization when expressed in other cells (see abstract by Zheng et al.). We are currently characterizing the thermotaxis behavior of these strains. Any thermotaxic phenotype observed in glr-5(ak57) may be a result of the inability of RIA to receive signals from the presynaptic cell AIY. Hobert et al. have demonstrated that the gene ttx-3 is expressed only in AIY. Using ttx-3::VAMP::CFP and glr-5::GLR-5::YFP constructs we are looking at the presynaptic and post-synaptic components of this synapse to determine if glr-5 could be mediating the signal from AIY.

Yingsong Hao et al. (2001) International C. elegans Meeting "USING YEAST-TWO HYBRID AND RNA-MEDIATED INTERFERENCE TO IDENTIFY PROTEINS ESSENTIAL FOR PIE-1 ACTIVITY AND LOCALIZATION IN EARLY EMBRYOS"

Geraldine Seydoux, Yingsong Hao

[International C. elegans Meeting, 2001]

The CCCH zinc finger protein PIE-1 is an essential regulator of germ cell fate that segregates with the germ lineage in early C. elegans embryos. PIE-1 has at least two functions in the embryonic germ lineage: 1) inhibition of mRNA transcription, and 2) promotion of efficient NOS-2 expression, a maternal protein required for primordial germ cell development. Structure-function studies have indicated that these two functions require sequences in the C-terminal region of PIE-1 (Tenenhaus et al., 2001). The C-terminus of PIE-1 is also required for asymmetric segregation of PIE-1 in dividing germline blastomeres (Reese et al., 2000). We have used the C-terminus of PIE-1 as a bait in a yeast two hybrid screen in the hope of identifying proteins required for PIE-1 activity and/or localization. Screening of 900,000 transformants yielded 365 candidate interactors (thanks to Zheng Zhou and Bob Horvitz for their excellent library). To quickly identify functionally relevant candidates, we designed a simple protocol to test the in vivo function of each candidate by RNAi. We first isolate the "prey" plasmids from the positive yeast transformants by selection on an auxotrophic E. coli strain. The prey inserts are then PCR amplified and transferred by recombinational cloning into Lisa Timmons and Andy Fire&#146;s feeding vector and transformed into E. coli strain HT115. Resulting transformants are used to "feed" three tester worm strains: 1) a MED-1:GFP line (thanks to Morris Maduro and Joel Rothman) to assay for defects in transcriptional repression, 2) a GFP: nos-2 3&#146;UTR line to assay for defects in NOS-2 expression, and 3) a PIE-1:GFP line to assay for defects in PIE-1 localization. The screen is ongoing and progress will be reported at the meeting.