Tong, Ada et al. (2013) International Worm Meeting "Exploring the role of rapsyn-1 in regulating C. elegans behavior."

Chalasani, Sreekanth, Tong, Ada

[International Worm Meeting, 2013]

Acetylcholine (ACh) signaling is a well-studied pathway in the neuromuscular junction (NMJ), but its role in the nervous system is poorly characterized. Rapsyn is a 43-kD protein that clusters and anchors acetylcholine receptors (AChR) in the postsynaptic membrane of the NMJ. Mutations in rapsyn have been associated with neuromuscular diseases in humans. For example, the N88K mutation in the RAPSN gene causes congenital myasthenic syndrome (1). In mice, targeted disruption of the RAPSN gene causes death within a few hours of birth (2). In C. elegans, however, rapsyn-1 (rpy-1) mutants are viable and appear wildtype in their locomotory ability. We plan to combine genetics and behavioral analysis to understand the role of rpy-1 in the worm. We find that rpy-1 mutants display a gain-of-function behavioral phenotype in the local search assay. They make twice as many turns in the first 15 minutes off food when compared to N2 wildtype animals. In order to test where rpy-1 functions, we performed knockdown experiments by expressing sense and antisense rpy-1 transcripts in muscles or in neurons. Surprisingly, neuronal knockdowns of rpy-1 are similar to rpy-1 mutants, while muscle knockdowns appear wildtype. This suggests that rpy-1 is required in the neurons rather than in the muscle. We will use transgenic rescue experiments to identify the cellular sites of rpy-1 action. We also plan on using candidate gene mutants and biochemical methods to identify components of the rpy-1 signaling pathway. These studies will provide insights into ACh signaling in the nervous system. (1) Dunne, V., Maselli, R. A., Hum. Genet. 49: 366-369 (2004) (2) Gautam, M., et al., Nature 377: 232-236 (1995).

Curran, Kevin et al. (2013) International Worm Meeting "Chemosensing a predator: Pristionchus pacificus and C. elegans."

Srinivasan, Jagan, Tong, Ada, Chalasani, Sreekanth, Joens, Matthew, Curran, Kevin, Fitzpatrick, James

[International Worm Meeting, 2013]

A current challenge in neuroscience is to bridge the connections between genes, neurons, neural circuits, and behavior in a single animal model. We are approaching this convergence by exploring the mechanisms governing a social interaction between a predator and its prey. A starving predator, Pristionchus pacificus, an omnivorous nematode, will attack and kill its prey, Caeonrhabditis elegans, when the two species share an agar plate. Certain Pristionchus strains exhibit carnivorous mouth morphology allowing them to slice open C. elegans cuticular exoskeleton and consume the underlying tissue. We have employed a novel imaging technique, Helium Ion Microscopy (HIM), to image the mouth cavities and teeth of several Pristionchus isolates. The tooth morphology revealed via HIM imaging correlates with observed feeding behaviors of the Pristionchus strains. We also find that, upon exposure to Pristionchus, C. elegans exhibit an increase in avoidance behavior. Moreover, we have identified ASI, ASJ and ASH as the specific chemosensory neurons that drive this avoidance behavior. We next performed mutant analysis coupled with cell specific rescue experiments and concluded that ocr-2, a TRPV channel, is required in ASH, while tax-4, a cGMP channel, is required in ASI and ASJ, in order to properly execute C. elegans predator avoidance behavior. Finally, this behavior is modulated by anti-anxiety drugs, suggesting that predator avoidance may serve as a model for complex human behaviors, such as fear and anxiety.

Liu, Zheng et al. (2015) International Worm Meeting "A redundant neural circuit drives C. elegans responses to social cues."

Tong, Ada, Leinwand, Sarah, Curran, Kevin, Chalasani, Sreekanth, Chute, Christopher, Liu, Zheng, Srinivasan, Jagan

[International Worm Meeting, 2015]

Animals communicate with each other using social cues. Typically, the sender sends a signal that modifies the current and/or future behaviors of the receiver. One particular form of communication that has been well studied is the interactions between a predator (sender) and prey (receiver). Despite these efforts, the neural circuits in the receiver that detect signals and driving behaviors remain poorly understood. We have developed a novel model of social behaviors analyzing the interactions between two nematodes P. pacificus (sender) and C. elegans (receiver).We find that C. elegans avoids secretions from a starving P. pacificus. We used cell ablations and calcium imaging to map the underlying sensory circuit. We find that either ASH and ASI or ASH and ASJ neurons detect P. pacificus derived signals. Moreover, we show that tax-4 (cyclic nucleotide gated ion channel) is required in both ASI and ASJ neurons while ocr-2 (TRP channel) is required in ASH for the avoidance response. We performed a pilot screen using small molecule drugs and identified Sertraline, (Zoloft, a selective serotonin reuptake inhibitor) as a candidate hit. Animals treated with Zoloft show greatly attenuated responses to P. pacificus. Surprisingly, its action requires GABA but not serotonin signaling to attenuate C. elegans avoidance. Apart from this rapid avoidance, we also show that prolonged exposure to Pristionchus secretions also modifies future C. elegans behaviors. These studies show how a receiver neural circuit detects social signals and modifies its current and future behaviors. .

Guenther C et al. (1995) International C. elegans Meeting "HAM-1 DISTRIBUTES DEVELOPMENTAL POTENTIAL DURING SEVERAL ASYMMETRIC CELL DIVISIONS"

Garriga G, Guenther C

[International C. elegans Meeting, 1995]

During C. elegans embryonic development, hundreds of asymmetric cell divisions generate cell diversity. To understand the molecular mechanisms responsible for establishing asymmetries during cell division, we have characterized mutations in the gene ham-1 that disrupt asymmetric cell divisions in five lineages: the RID, ADE/ADA, ADL, HSN/PHB and ALN/PLM lineages. Based on our findings, we propose a model in which ham-1 acts prior to cell division to segregate factors that confer specific fates to daughter cells. Immunocytochemistry has shown that the neurons generated by the affected lineages can be missing, present in the normal numbers or duplicated in ham-1 mutants. Since each affected lineage normally produces one cell that dies, we analyzed ham-1 ced-3 double mutants to determine if the missing cells in ham-1 were caused by cell death. All of the affected lineages including those that did not produce extra neurons in ced-3 mutants always produced extra neurons in ham-1 ced-3 mutants. Therefore, production of extra cells in ham-1 mutants is often masked by cell death.

Chuang, Marian et al. (2015) International Worm Meeting "Death Associated Protein Kinase (DAPK-1) interacts with microtubule dynamics regulators in epidermal development and wound repair."

Chuang, Marian, Chisholm, Andrew, Tong, Amy, Hsiao, Tiffany

[International Worm Meeting, 2015]

Wounding the C. elegans epidermis triggers innate immune and cytoskeletal responses that collaborate to allow animals to survive otherwise lethal damage. In C. elegans the death-associated protein kinase ortholog DAPK-1 is required for normal epidermal morphology and negatively regulates epidermal wound responses (Tong et al., 2009, PNAS). dapk-1 mutants display hyperactive wound responses, including faster wound closure and constitutive expression of antimicrobial peptides. To further understand how DAPK-1 regulates these processes we screened for suppressors of the dapk-1 morphological phenotypes and identified mutations in several known microtubule (MT) regulators. One suppressor causes complete loss of function in ptrn-1, which encodes the C. elegans member of the Patronin/Nezha/CAMSAP family of microtubule (MT) minus-end binding proteins (Chuang et al., 2014, Cell Reports). We find that PTRN-1 is required cell autonomously in the epidermis to alter dapk-1 phenotypes. ptrn-1 null mutants display normal epidermal development but are defective in wound repair. We present genetic and pharmacological evidence suggesting that DAPK-1 regulates epidermal microtubule stability. Furthermore, preliminary studies suggest DAPK-1 itself is also transported within the epidermis. We are using quantitative particle tracking analysis to characterize DAPK-1 movement and its relationship to the epidermal MT cytoskeleton. Our work reveals novel roles for DAPK-1 and microtubule (MT) regulators in the development and function of the mature epidermis.

Amy Tong et al. (2005) International Worm Meeting "The role of C. elegans DAP kinase MOR-3 in post-embryonic epidermal development"

Andrew CHISHOLM, Amy Tong

[International Worm Meeting, 2005]

Mutations in mor-3 cause an unusual late-onset epidermal morphogenesis defect (Tong et al, 2004 WCWM abstract 193). mor-3 mutants display vacuolation, thickening, and encrustation of specific regions of the epidermis (notably the nose, dorsal midline, vulva, and tail) that become apparent from later larval stages onwards. Using EM we have found that the cuticle in such regions is up to 4 times thicker than normal and has an aberrant ultrastructure. Markers for the cuticle cortex (COL-19::GFP) and struts (BLI-1::GFP) show mislocalization and accumulation in mor-3 mutants. Speculatively, MOR-3 might act in negative feedback control of cuticle secretion in the epidermis. mor-3 encodes the C. elegansortholog of death-associated protein kinase (DAP kinase, DAPK), a protein previously implicated in apoptotic and autophagic cell death. Preliminary analysis indicates that the epidermal defects in mor-3 mutants do not reflect aberrant activation of known cell death pathways. However mor-3 mutants do have subtle defects in apoptosis (Yi-Chun Wu, personal communication) suggesting that CeDAPK/MOR-3 may have separable roles in apoptosis and epidermal development. A functional MOR-3::GFP is first detectable in the epidermis in late embryonic stages. In larvae and adults MOR-3::GFP is punctate within epidermal cells. We are trying to determine the identity of these puncta. MOR-3 reporter genes are also highly expressed in body wall and vulval muscles and in some neurons; however, genetic mosaic analysis suggests mor-3 function in muscles is not required for epidermal morphogenesis. We are using tissue specific promoters to define when and where MOR-3 function is required in epidermal development. We are also trying to determine which domains of MOR-3 are important in its epidermal function, and to identify interacting partners of MOR-3 using genetic modifier screens.

Brendan Mattingly et al. (2006) Development & Evolution Meeting "EXC-5: Keeping Worm Tubes in Shape"

Brendan Mattingly, Matthew Buechner, Michael Peterson

[Development & Evolution Meeting, 2006]

The C. elegans excretory system consists primarily of a large, tubular, H-shaped structure made from a single excretory canal cell. This cell, located near the terminal bulb of the pharynx, extends two hollow processes on either side of the animal to form two excretory canals that extend to the head and tail on both the left and right sides of the animal. The EXC-5 protein is homologous to a human guanine nucleotide exchange factor (GEF) FGD1, which regulates formation of facial, genital, and limb structures. Null mutations in exc-5 cause defects in lumen diameter in the form of large fluid-filled cysts, which in the worst cases can become as large as the entire body diameter of the worm. We believe these cysts form as a result of a mechanical failure of the actin cytoskeleton lining the apical surface of the canal. Canal extension during development generally ceases at the point where very large cysts form. Recently, we performed an EMS non-complementation screen to identify new mutations in the exc-5 gene. We have recovered 42 isolates. Two of these, qp21 and qp39, have been sequenced, and identified as having point mutations resulting in premature stop codons in the C-terminal Pleckstrin-Homology domain of the protein. The phenotype of these alleles is similar to the null-phenotype of the rh232 deletion. This screen also produced several mutations that exhibit hypomorphic and temperature-sensitive defects in canal formation. In addition to our allelic analysis, we are currently investigating the effects of cdc-42 on canal formation and possible interactions between cdc-42 and exc-5, and between exc-5 and exc-9 (see abstract by X. Tong et al.). Preliminary data indicate that exc-5 is necessary for both extension and maintenance of the canals. Finally, in order to identify additional genetic interactions with exc-5, we are developing a method to use RNAi solely in the excretory canals, in order to look at the effects of a canal-specific knockout of cdc-42 and other genes.

Shaye, Daniel et al. (2013) International Worm Meeting "Excretory canal development requires conserved kinases and exc-6/INF2, a formin implicated in kidney disease."

Shaye, Daniel, Greenwald, Iva

[International Worm Meeting, 2013]

The excretory canal cell, which is required for osmoregulation, is a simple model to study tubulogenesis. It extends long processes first dorsally, then anterior and posteriorly, each with an intra-cellular lumen. Previous genetic analysis revealed that canal outgrowth requires some genes that also mediate neuronal outgrowth1-3; lumen formation is driven by regulation of fluid and ion transport 4,5,6; and lumen maintenance depends on the apical actin cytoskeleton7,8. To identify new conserved genes involved in tubulogenesis, we used feeding RNAi in a sensitized background to test 243 conserved kinases9, identifying 9 that caused excretory canal phenotypes. We have initially focused on the kinase PIG-1/MELK. Loss of pig-1 causes shortening of anterior and posterior canal arms, a mild cystic phenotype and regions with multiple lumens, similar to phenotypes seen in the uncloned mutant exc-610. We cloned exc-6, and found that it encodes an ortholog of the human formin INF2, a kidney disease gene11. INF2 promotes actin polymerization, de-polymerization and microtubule stability12. We have evidence that residues that regulate actin dynamics are required for EXC-6 function. Unexpectedly, EXC-6 may not predominantly co-localize with apical actin, instead accumulating with structures resembling microtubules. Thus, EXC-6 may coordinate actin and microtubule cytoskeletons in the excretory canal. Disease-causing INF2 mutations are dominant and have been proposed to result in constitutive activity. Using rescue of exc-6(0), we have found functional evidence supporting this model. We are currently investigating the functional relationship between pig-1 and exc-6 in canal development. Given the conservation of INF2 function suggested by our rescue experiment, such a relationship might have potential relevance for disease. 1) Stringham et al., 2002. 2) Katidou et al., 2012. 3) Marcus-Gueret et al., 2012. 4) Khan et al., 2013. 5) Kolotuev et al., 2013. 6) Berry et al., 2003. 7) Tong and Buechner, 2008. 8)Buechner, 2002. 9) Shaye and Greenwald, 2011. 10) Buechner et al. 1999. 11) Brown et al. 2010. 12) Gaillard et al., 2011.

Tong, Yong Guang et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A regulatory network of homeobox genes is required for the function of the Caenorhabditis elegans excretory cell"

Tong, Yong Guang, Meemon, Krai, Burglin, Thomas

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Homeobox genes play important roles in the development and differentiation of animals. We have previously found that the POU-III class homeobox gene, ceh-6, functions in the excretory cell (Brglin et al. 2001). We have been studying the three C. elegans otd/Otx homeobox genes, ceh-36, ceh-37 and ttx-1 (Tong et al., IWM 2007). Using mutant alleles and RNAi we have found that ceh-37 and ttx-1 act redundantly in the excretory cell. Double mutants arrest with excretory cell defects. Furthermore, we have found that the prospero homeobox gene ceh-26 is expressed in the excretory cell and loss of function of ceh-26 causes early larval lethality consistent with excretory cell defects. We further examined the effects of knocking down ceh-6, ceh-26, and ceh-37/ttx-1 in combination by looking at GFP reporters expressed in the excretory cell. For example, knock-down of all genes downregulates the expression of clh-4::GFP in the excretory cell. In contrast, expression of sulp-4::GFP is suppressed in ceh-26(RNAi) and ceh-6(mg6) animals, but not in ceh-37(RNAi)/ttx-1(RNAi) animals. Further, only ceh-6(RNAi) completely abolishes GFP expression of pgp-12, but ceh-26(RNAi) and ceh-37(RNAi)/ttx-1(RNAi) only weakly reduce the level of GFP. We also investigated the regulation of the homeobox genes amongst each other using RNAi and homeobox::GFP integrated lines. ceh-6(RNAi) nearly totally suppresses the GFP expression of both ceh-37 and ceh-26. ceh-26 (RNAi) also abolishes ceh-37::GFP expression. ceh-37/ttx-1(RNAi) of ceh-26::GFP showed that 57.78% of the arrested worms still had GFP expression, indicating a partial downregulation, possibly a r egulatory feedback loop. Based on our findings, we propose a regulatory hierarchy with ceh-6 at the top, ceh-26 in the middle, and the otd/Otx genes downstream. Yet, not all excretory cell genes depend on this ceh-6 cascade, suggesting the other factors remain to be uncovered.

Tong, Yong-Guang et al. (2011) International Worm Meeting "A regulatory network of homeobox genes is required for the function of the Caenorhabditis elegans excretory cell."

Tong, Yong-Guang, Meemon, Krai, Burglin, Thomas R.

[International Worm Meeting, 2011]

Homeobox genes play important roles in the development and differentiation of animals. We have previously found that the POU-III class homeobox gene, ceh-6, functions in the excretory cell (Burglin et al. 2001). We have been studying the three C. elegans otd/Otx homeobox genes, ceh-36, ceh-37 and ttx-1 (Tong et al., IWM 2007). Using mutant alleles and RNAi we have found that ceh-37 and ttx-1 act redundantly in the excretory cell (Meemon et al., EWM 2008). Double mutants arrest with excretory cell defects. Furthermore, we have found that the prospero homeobox gene ceh-26 is expressed in the excretory cell and loss of function of ceh-26 causes early larval lethality consistent with excretory cell defects. We further examined the effects of knocking down ceh-6, ceh-26, and ceh-37/ttx-1 in combination by looking at GFP reporters expressed in the excretory cell. For example, knock-down of all genes downregulates the expression of clh-4::GFP in the excretory cell. In contrast, expression of sulp-4::GFP is suppressed in ceh-26(RNAi) and ceh-6(mg6) animals, but not in ceh-37(RNAi)/ttx-1(RNAi) animals. Further, only ceh-6(RNAi) completely abolishes GFP expression of pgp-12, but ceh-26(RNAi) and ceh-37(RNAi)/ttx-1(RNAi) only weakly reduce the level of GFP. We also investigated the regulation of the homeobox genes amongst each other using RNAi and homeobox::GFP integrated lines. ceh-6(RNAi) nearly totally suppresses the GFP expression of both ceh-37 and ceh-26. ceh-26 (RNAi) also abolishes ceh-37::GFP expression. ceh-37/ttx-1(RNAi) of ceh-26::GFP showed that 42%; of the arrested worms still had GFP expression, indicating a partial downregulation, possibly a regulatory feedback loop. Based on our findings, we propose a regulatory hierarchy with ceh-6 at the top, ceh-26 in the middle, and the otd/Otx genes downstream. Yet, not all excretory cell genes depend on this ceh-6 cascade, suggesting the other factors remain to be uncovered.