Chou, Ting-Chen et al. (2011) International Worm Meeting "Characterization of the pathogenicity of Enterohaemorrhagic E. coli in C. elegans."

Chiu, Hao-Chieh, Chou, Ting-Chen, Chen, Chang-Shi, Wu, Ching-Ming, Kuo, Cheng-Ju

[International Worm Meeting, 2011]

Strains of Enterohaemorrhagic Escherichia coli (EHEC) that produce verocytotoxin, such as E. coli O157:H7, form an important group of zoonotic pathogens. E. coli O157:H7 has been implicated in large outbreaks as well as in sporadic cases of haemorrhagic colitis and the sometimes fatal haemolytic uremic syndrome. Here we applied the genetic tractable animal, C. elegans, as a model host to study the virulence of EHEC as well as the host innate immunity to this pathogen. Our preliminary data showed that EHEC intoxicated, induced severe Bag (bag-of-worms adults) phenotype, and eventually killed the worms. The transmission electron microscopy results indicated that EHEC induced attaching and effacing (A/E) lesions in the intestine. By further genetic analysis, we found that some important virulence factors of EHEC were required for its toxicity in C. elegans, and some conserved innate immune signal pathways were mediated in host susceptibility. Our results suggest that this EHEC-C. elegans model is suitable for future comprehensive genetic screens in both bacterial and host factors involved in the pathogenesis of EHEC.

Kuo, Cheng-Ju et al. (2013) International Worm Meeting "Shiga-like toxin 1 confers the full pathogenicity of Enterohaemorrhagic Escherichia coli and activation of the p38/MAPK pathway in Caenorhabditis elegans."

Chiu, Wen-Tai, Chen, Chang-Shi, Chiu, Hao-Chieh, Kuo, Cheng-Ju, Syu, Wan-Jr, Chou, Ting-Chen

[International Worm Meeting, 2013]

Enterohaemorrhagic Escherichia coli (EHEC), a major foodborne pathogen, is responsible for life-threatening diseases in humans as a consequence of the production of Shiga-like toxins. Currently, there is no specific treatment for EHEC infection and the use of antibiotics is contraindicated. Moreover, lack of a good animal model system hinders the study of EHEC virulence with systematic methods in vivo. To these regards, we applied the genetic tractable animal model, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host innate immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. We also demonstrate that E. coli O157:H7 colonizes and replicates in C. elegans, stimulates the ectopic expression of microvillar actin, and induces the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium in vivo. Moreover, our genetic analyses corroborate that the Shiga-like toxin 1 (Stx1) of E. coli O157:H7 is required partly for its toxicity. The C. elegans p38 MAP kinase signaling pathways, an evolutionally conserved innate immune signaling pathways, are activated and mediated in the regulation of host susceptibility to EHEC infection in a Stx1-dependent manner. Given that the bacterivore C. elegans and bacterium, in this case E. coli, may have generated a diverse arsenal for both parties to defend each other during the long-term evolution of this predator-prey relationship, our results suggest that this EHEC-C. elegans model is suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.

Catherine Chiu et al. (2002) West Coast Worm Meeting "Three miscellaneous axon guidance genes"

Ray Squires, Scott G Clark, Catherine Chiu

[West Coast Worm Meeting, 2002]

To understand further the molecular mechanisms involved in growth cone guidance, we performed several screens to identify new genes involved in axon extension and pathfinding. Mutants with a variety of axon outgrowth, branching and pathfinding defects as well as mutants with either axonal degeneration, cell fate, cell migration, cell position or programmed cell death defects were recovered. Over 160 mutants were isolated that define over 40 genes (see abstract by Chiu, Sabater, Squires & Clark). We cloned five of these genes: zag-1, tba-1, klp-7, mig-11 and wly-1.

Motegi, Fumio et al. (2009) International Worm Meeting "PAR-2 is a microtubule-binding protein that links microtubules and cortical polarity in C. elegans zygotes."

Motegi, Fumio, Zonies, Seth, Seydoux, Geraldine

[International Worm Meeting, 2009]

The C. elegans zygote becomes polarized shortly after fertilization by the sperm-donated microtubule-organizing center (MTOC), which induces PAR polarity on the cortex. How the MTOC communicates with the cortex is not known. Possible signals include proteins that accumulate on the MTOC''s pericentriolar material (PCM) and the microtubules themselves (Motegi and Seydoux, 2007 for review). We have obtained evidence for a direct role for microtubules by studying PAR-2. PAR-2 is a RING finger domain protein that accumulates on the cortex nearest the MTOC during polarization (Boyd et al, 1996). PAR-2 also accumulates on the MTOC itself, most prominently during mitosis. We have found that PAR-2 has a strong affinity for microtubules in vitro (KDapp<0.2 mM). This affinity depends on three separate microtubule-binding domains, which overlap with the domain in PAR-2 required for localization to the cortex (Hao et al., 2006). Mutations in one of the domains reduce PAR-2 affinity for microtubules by 5 fold. When expressed in zygotes depleted of endogenous PAR-2, this PAR-2 mutant was defective in localizing to MTOCs, but was able to localize to the posterior cortex and rescue the par-2 embryonic lethality as efficiently as wild-type, suggesting that microtubule binding is not essential under normal conditions. The microtubule-binding defective mutant, however, was not able to localize to the cortex in zygotes that lack or delay PCM assembly or have reduced cortical actin contractility. In those zygotes, wild-type PAR-2 can still accumulate on the cortex nearest the MTOC, but PAR-2 defective in microtubule binding cannot and remains in the cytoplasm. We conclude that microtubule binding contributes to PAR-2 localization under conditions when the MTOC signal is compromised. Along with previous findings, these data suggest that polarity establishment in the zygote depends on two redundant mechanisms: a first mechanism dependent on efficient PCM assembly and actin contractility, and a second mechanism dependent on microtubules and PAR-2. Boyd, L., Guo, S., Levitan, D., Stinchcomb, D.T., Kemphues, K.J. (1996). Development 122, 3075-3084. Hao, Y., Boyd, L., Seydoux, G. (2006). Developmental Cell 10, 199-208. Motegi, F., Seydoux, G. (2007). JCB 179, 367-369.

D Fitch et al. (1999) International C. elegans Meeting "Evolution of Rhabditidae, a family for C. elegans"

M Sutherlin, C Nguyen, P De Ley, A Vierstraete, D Fitch, K Kiontke, W Sudhaus, J Vanfleteren, R De Wachter, N Eakin

[International C. elegans Meeting, 1999]

C. elegans provides a model for discovering how genes produce form. Applying this model to discern how changes in these genes have produced different forms first requires knowledge about the evolution of species closely related to C. elegans . We have traced the evolution of some important features such as vulval and male tail pattern and morphogenesis, characteristics of the stoma and gonads, hermaphroditism, and copulatory behavior. To trace these evolutionary changes, we have inferred phylogenetic relationships among the major species groups in family Rhabditidae, of which C. elegans is a member. Our data show that the vulva was originally located midbody and posteriad VPC migrations occurred independently at least twice, correlating with changes to one-armed gonads. In the male tail, some rays have independently changed positions, suggesting a degree of mosaicism, whereas some seam cells share fate changes, consistent with coordinate regulation. Male tail tip morphogenesis has undergone several changes between "leptoderan" and "peloderan" in different ways (e.g., peloderan tails have been formed by tail hypodermis retraction with or without cell fusion, revealing separable mechanisms). Both losses and gains of coiling to stabilize copulation have occurred, correlating with some aspects of male tail form. The glottoid part of the stoma has been reduced or lost more than once, explaining a difference from previous phylogenetic hypotheses which assumed absence is primitive. Hermaphrodites derived from females in independent lineages, but no unambiguous case exists for the reverse. Relationships were inferred from nearly complete 18S ribosomal RNA genes aligned by modeling secondary structures. Although our data show that some intraspecific polymorphism exists, most relationships are well resolved. Increasing taxon representation (50 Rhabditidae) substantially improves the resolution. Other contributors: C. Pomilla and J. Chiu (New York University) and A. Duhachek (University of Nebraska).

Lung Yam et al. (2001) International C. elegans Meeting "zd8 causes cell migration and axon fasciculation defects"

Scott Clark, Lung Yam

[International C. elegans Meeting, 2001]

Neurons project axons during development that migrate long distances along stereotypic pathways to find their appropriate targets and establish the initial connectivity of the nervous system. The trajectory of each axon is determined by the response of its growth cone to spatial signals along its route. These signals include cell surface and extracellular matrix molecules that provide short-range cues as well as diffusible molecules that provide long-range guidance information. These signals can either attract or repel a migrating growth cone and, through their combined action, these signals orchestrate correct axon outgrowth and pathfinding. To identify genes involved in axon growth and guidance, we have performed several screens for mutants with axon pathfinding defects (see abstract by Chiu et al.). One mutant identified in our screens, zd8 , has a variety of cell migration and axon guidance defects. zd8 mutants are uncoordinated and have a notched head and a withered tail phenotype. The PVQ neurons are located in the tail and each projects a single axon that runs in the ventral nerve cord to the nerve ring in wild type animals. The PVQ axons often defasciculate from the nerve cord and wander anteriorly in lateral positions in zd8 mutants. We also found defects in the migration and axon outgrowth of the HSNs. The HSNs are often found either anterior or posterior of their normal position in the midbody and their axons often fail to follow their normal trajectory to the nerve ring in zd8 animals. zd8 also causes defects in the growth of other axons. Thus, the gene defined by zd8 is needed for both cell migration and axon fasciculation. To clone and characterize further this gene, we mapped zd8 to a small interval and are testing cosmids in this region for rescue.

Zou, Y. et al. (2019) International Worm Meeting "Dendritic transport of KPC-1 transcripts mediates local dendrite self-avoidance of a nociceptive neuron."

Zou, W., Shih, M., Chuang, C.-F., Chiu, H., Ferreira, T., Shen, K., Chang, C., Zou, Y., Suzuki, N.

[International Worm Meeting, 2019]

The elaborate branching patterns displayed by PVD neurons in C. elegans are constrained by the organizing principle of self-avoidance. Self-avoidance is a process in which self-recognition among neurites of a single neuron is followed by their repulsion from each other. This selective repulsion between dendrites of the same cell was originally observed in leech neurons to promote a uniform receptive field. Recent studies by Zipursky and colleagues using Drosophila as a model found that homophilic binding of Dscam1 proteins on sister branches of the same cell (MB or da neurons) promotes repulsive interactions between them, to ensure that they diverge and grow along separate pathways. Since the Dscam1 mutation does not completely disrupt dendrite self-avoidance in Drosophila and there is no Dscam1 homolog in C. elegans, studying PVD dendrite self-avoidance in C. elegans provides a unique opportunity to identify novel mechanisms of dendrite self-avoidance. We isolated mutants through genetic screens that displayed dendrite self-avoidance defects in PVD neurons. We identified mutations in the kpc-1 gene as being responsible for this defective self-avoidance phenotype through whole genome sequencing. kpc-1 mutants displayed a significantly lower tertiary branch avoidance index and decreased sensory coverage in the skin compared to wild-type animals. KPC-1 encodes a proprotein convertase subtilisin/kexin (PCSK). Although kpc-1 has been implicated in dendrite self-avoidance and arborizationa, the mechanism by which kpc-1 regulates this process is unknown. Here, we show that kpc-1 functions in PVD neurons to promote dendrite self-avoidance in a kpc-1 3'UTR dependent manner. We are currently developing an in vivo imaging system to visualize kpc-1 RNA localization in PVD dendrites and to determine the role of kpc-1 local translation in dendrite self-avoidance. Reference: a. Dong X, Chiu H, Park YJ, Zou W, Zou Y, Ozkan E, Chang C*, Shen K*. (2016) "Precise regulation of the guidance receptor DMA-1 by KPC-1/Furin instructs dendritic branching decisions," eLife, 5, e11008. *Co-corresponding authors

Raut S V et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "UNC-86, a POU Homeodomain Protein, Regulates Expression of hlh-3 in C. elegans"

Raut S V, Alfonso A

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

HLH-3 is a basic helix-loop-helix (bHLH) transcription factor of the Achaete-Scute family that plays a role in the differentiation of the serotonergic hermaphrodite specific motor neurons (HSNs) in C. elegans. hlh-3(bc248) animals are axonal pathfinding defective and 65% do not show immunoreactivity to anti serotonin (5HT) antibodies (Doonan et al.,2008). The non immunoreactive HSNs do not undergo apoptosis, neither do they change their fate to their sister cells (PHBs). These HSNs are born and migrate to their proper location but are apparently non functional based on pharmacological assays. They seem to stall in their differentiation program (Doonan et al.,2008). There are other transcription factors like unc-86, zag-1, sem-4 that also play a role in specifying the serotonergic phenotype of the HSNs (Clark &amp; Chiu, 2003; Sze, Zhang, Li, &amp; Ruvkun, 2002; Wacker, Schwarz, Hedgecock, &amp; Hutter, 2003). To address the possibility that hlh-3 is regulated by one of these transcription factors we used a GFP reporter transgene fused to a hlh-3 genomic fragment in different mutant backgrounds. We saw no change in hlh-3 expression in the HSNs in zag-1 and sem-4 mutant animals. However, unc-86 mutant animals showed absence of hlh-3 expression in 75% of the HSNs. UNC-86 is a POU homeodomain protein that is responsible for late differentiation of HSNs. In unc-86 mutant animals, HSNs are non serotonergic and we will show that they have an axonal pathfinding defect. Given that there are potential POU binding sites in the hlh-3 promoter, we hypothesize that UNC-86 directly regulates hlh-3 expression by binding to these sites. In the future we intend to mutagenize these sites by site-directed mutagenesis and analyze the expression of hlh-3. We will also do serial promoter deletions of hlh-3 to identify the minimal promoter region necessary for expression.

Ahmed Mohamed et al. (2005) International Worm Meeting "The VAB-1 Eph RTK is Required for Proper Axon Guidance and Neuronal Cell Positioning."

Ian D Chin-Sang, Ahmed Mohamed

[International Worm Meeting, 2005]

The vertebrate Eph receptor tyrosine kinases (RTKs) and their ligands, the Ephrins, have been found to have diverse developmental roles including early aspects of embryogenesis, segmental boundary formation, cell migration, neurogenesis, axon guidance, dendritic spine morphogenesis and tumorigenesis. Mutations that affect the only C. elegans Eph RTK, VAB-1, cause defects in neuroblast migration and epidermal morphogenesis. Recent work has also revealed the presence of some weak neuronal defects that include aberrant axon crossing in the ventral midline, disruption in the ventral guidance of amphid axons, anterior axon outgrowth in the nerve ring, and ipsilateral outgrowth of pharyngeal neurons (Bulow and Hobert, Neuron 2004 41:723-36; Hao et al., Nueron 2001 32:25-38; Morck et al., Developmental Biology 2003 260:158-75; Zallen et al., Development 1999 126:3679-92). To further understand the role of VAB-1 in neuronal development, we characterized the neuronal and behavioral phenotypes of both loss-of-function and gain-of-function VAB-1 Eph signaling in neurons. We specifically analyzed the PLM mechanosensory neuron, the canal-associated neurons (CANs) and the DVB GABAergic neuron. We found that vab-1 mutants had weak neuronal defects where axons overextended beyond their usual target region. To investigate the effects of a VAB-1 gain-of-function, we created a hyper-active form of VAB-1 by targeting the intracellular portion of VAB-1 to the plasma membrane via N-terminal myristoylation (MYR). The MYR-VAB-1 resulted in a constitutively active tyrsoine kinase and induced a penetrant phenotype where axons terminated prematurely before reaching their target, a phenotype opposite of the vab-1 loss-of-function mutants. Our results suggest that the VAB-1 Eph RTK is required for targeting or limiting axons and neuronal cells to a specific region perhaps by transducing a repellent or stop cue.

Boxem, Mike (2009) International Worm Meeting "Mapping the protein interaction network that controls cell polarity."

Boxem, Mike

[International Worm Meeting, 2009]

Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .