Ramirez-Fajardo, Emilio et al. (2021) International Worm Meeting "Innate immune responses of Caenorhabditis elegans to the emerging pathogen Elizabethkingia anophelis"

Schmidt, Kristopher, Albayati, Wesam, List, Skylar, Ramirez-Fajardo, Emilio, Grossen, Sarah, Siderhurst, Matthew

[International Worm Meeting, 2021]

Elizabethkingia anophelis (Ea) is an emerging pathogen that causes sepsis, meningitis, and death in people with comorbidities. While usually innocuous, a recent community outbreak resulted in at least 20 deaths and 66 laboratory confirmed cases. The outbreak stemmed from the rapid formation of a novel and more virulent Ea sublineage created by a conjugative element that disrupted the muty DNA repair gene. The virulence associated factors, pathogenic mechanisms, and ecology of Ea are not known. C. elegans is a well-established model for the characterization of pathogen-host interactions. When we exposed mixed stage wild-type C. elegans to Ea (Ag1 strain), the animals exhibited decreased movement, slowed development, early larval death, and an overall decreased fecundity compared to animals exposed to E. coli OP50 (Ec). We also made the striking observation that worms exposed to Ea actively avoided Ea lawns. To understand the aversive response, we conducted two-choice assays between Ea and Ec in experiments using L1 and L4 animals. L1s not previously exposed to a food source initially chose Ea over Ec (-0.75 CI, Choice Index [CI] of -1.0 represents Ea selection) but actively selected Ec over time (+0.41 CI at 96hrs). L4 animals first reared on Ec chose Ec over Ea in two-choice assays (+0.49 CI). Importantly, we found that staged adults placed on Ea bacteria without another bacterial food source adopted a lawn avoidance phenotype, suggesting that they are repulsed from Ea even in the absence of Ec. These avoidance responses depend on an intact odorant detection system because lim-4(ky403) and tax-2(p671); tax-4(p678) mutant animals failed to avoid Ea lawns. We used solid-phase microextraction (SPME) gas chromatography mass spectrometry (GC-MS) to identify volatile odorant differences between Ea and Ec and observed a marked increase in the amount of indole in Ec compared to Ea. Indole is an attractive compound recently shown to modulate predator-prey interactions between C. elegans and bacteria. To examine the pathogenic mechanisms of Ea in C. elegans, we exposed N2, sek-1 (km4), tol-1(nr2033), dbl-1(nk3) and daf-2(e1370) animals to Ea and Ec. Wild-type animals displayed several pathological changes when grown on Ea and died faster than their Ec exposed counterparts. Decreased lifespan and increased pathologies were found in sek-1 while daf-2 animals appeared immune to Ea. The tol-1 and dbl-1 alleles did not influence Ea pathogenicity. To complement these immunity assays we also examined the antimicrobial activity of dod-24, irg-1 and T24B8.5 in C. elegans exposed to Ea using transcriptional reporter GFP strains. We observed decreases in the expression of T24B8.5 and increased but more variable changes in irg-1. Taken together these experiments suggest that Ea is pathogenic to C. elegans, opening new opportunities to investigate pathogen and host factors influencing Ea related disease.

Theresa M. Grana et al. (2007) International Worm Meeting "Cell-cell adhesion molecules regulate gastrulation in C. elegans."

Theresa M. Grana, Elisabeth A. Cox, Jeff Hardin

[International Worm Meeting, 2007]

Morphogenesis requires dynamic regulation of cell adhesion and the cytoskeleton. The first major morphogenetic movement in development is gastrulation. C. elegans gastrulation begins at the 26-cell stage with the migration of the two endodermal precursors Ea/Ep from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on their specification and polarization, apical myosin accumulation and Wnt activated actin-myosin contraction that drives apical constriction and ingression (Nance et al., Wormbook, 2005; Lee et al., Current Biology, 2006). Here, we show that Ea/Ep ingression also requires cell-cell adhesion mediated by either HMR-1/cadherin or SAX-7/L1 CAM (L1 cell adhesion molecule). Either adhesion system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. In sax-7(eq1); hmr-1(RNAi) embryos we observe correct specification of Ea/Ep, based on timing of cell divisions and the presence of birefringent gut granules, and correct apical polarization of Ea/Ep, based on basolateral localization of PAR-2::GFP. Significantly, in sax-7(eq1); hmr-1(RNAi) embryos Ea/Ep fail to accumulate myosin (NMY-2::GFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) alone, apical myosin accumulation is equivalent to wildtype. (In gastrulation stage embryos, HMR-1 (as indicated by HMP-2 staining) and SAX-7 are localized at all sites of cell-cell contact and do not appear on exposed cell surfaces.) SAX-7 and HMR-1 may function to localize myosin via signaling or by directing localization of myosin-binding molecules.

Jen-Yi Lee et al. (2003) International Worm Meeting "The role of cell cycle timing in C. elegans gastrulation movements."

Bob Goldstein, Jen-Yi Lee

[International Worm Meeting, 2003]

Cytoskeletal machinery is used for both cell division and cell movement. For this reason,the cell cycle must be finely coordinated with motility. However, how this essential coordination is controlled remains unknown. During C. elegans gastrulation, two endodermal precursors, Ea and Ep, move into the center of the embryo and have a longer cell cycle than most of the cells at the same stage. Precocious Ea/p division in various mutant backgrounds is associated with gastrulation defects. However, a direct, causative relationship between cell cycle length and gastrulation has not been established. We asked if one of these genes, gad-1 (Knight and Wood, 1998), was specifically required for gastrulation movements. By delaying Ea/p division with a laser, we were able to rescue ingression of Ea/p in gad-1 mutant embryos, suggesting that gad-1 indirectly causes gastrulation defects by affecting the timing of Ea/p division.We next asked whether or not a longer cell cycle was sufficient for ingression in other pairs of cells by laser-delaying their divisions. Surprisingly, we found that pairs of ABxx, MSxx, and Cxx cells were able to ingress given a longer cell cycle. This suggested that Ea/p ingress by virtue of their long cell cycle period, whereas other cells do not ingress due to shorter cell cycle periods. We are currently investigating the molecular mechanisms behind cell cycle control of ingression. One possible model is that a certain length of time in a specific phase of the cell cycle is required for gastrulation, and that laser treatment pauses the cells in that specific phase. Using pharmacological inhibitors of the cell cycle, as well as cell cycle mutants, we are attempting to identify if elongation of specific cell cycle phases is sufficient to rescue gastrulation in gad-1 embryos. A second possible mechanism is that long cell cycle periods may allow an accumulation of proteins required for movement. We previously showed that ingression is driven by an actomyosin-based contraction of the apical side of Ea/p (Lee and Goldstein, 2003). Therefore, an attractive model is one whereby long cell cycle periods allow for the accumulation of molecules required for contractile forces at the apical surface of cells. One potential player we are currently examining with immunostaining is NMY-2, which was previously shown to localize and accumulate at the apical surface of Ea/p (Nance and Priess, 2002). Given the genetic, molecular, and embryological tools available for C. elegans, we hope to gain insight into the mechanisms through which the cell cycle controls cell movement.

Daniel J Marston et al. (2005) International Worm Meeting "Investigation of a role for cadherin-domain containing proteins in C. elegans gastrulation."

Daniel J Marston, Bob Goldstein

[International Worm Meeting, 2005]

In the C. elegans embryo gastrulation begins at the 26-cell stage with the migration of the two endoderm precursor cells, Ea and Ep, from the surface of the embryo to the embryonic interior. The cellular process driving this ingression of the Ea and Ep cells is an actin-myosin dependent constriction of the apical surface of these cells. Comparison with other systems would suggest that in order for this apical constriction to lead to ingression it is essential that cell-cell adhesion be maintained between the Ea and Ep cells, and/or the surrounding cells during the constriction. Supporting this is the observation that the surrounding cells are in close contact with the ingressing cells and they move round to cover the Ea and Ep cells as those cells move in to the embryo. There are more than 35 genes in the C. elegans genome thought to encode for adhesion proteins comprising several different classes, including cadherin proteins and IgCAMs. Currently there are no reported defects in gastrulation in strains carrying mutations in these genes. Furthermore when we used feeding RNAi to disrupt the function of those genes for which there is no mutant we detected no defects in gastrulation. One hypothesis is that there may be redundancy between multiple adhesion proteins. One way to overcome the challenge presented by redundancy is to inhibit the function of multiple adhesion proteins simultaneously. It is possible do this by microinjecting adult hermaphrodites with small pools of dsRNAs that code for different proteins. The adults then lay embryos with reduced expression of the proteins coded for by the pooled dsRNAs. Using this technique we have attempted to disrupt the function of multiple proteins that contain cadherin domains. We have identified a group of these proteins that are required for normal embryonic development. Furthermore we have been able to use 4D microscopy to follow cell migrations in embryos dissected out of the dsRNA treated adults to investigate the effects of reducing the function of these cadherin-domain containing proteins on cell movements during gastrulation.

Martijn Dekkers et al. (2003) International Worm Meeting "Genetic analysis and molecular imaging of olfactory signalling in C. elegans"

Martijn Dekkers, Gert Jansen, Gerard Borst

[International Worm Meeting, 2003]

G-protein signal transduction is one of the most widely used mechanisms for cells to communicate with their environment. In a single cell several different G-protein signalling cascades are present, and recent evidence suggests extensive crosstalk between these individual cascades. How this is organised, while maintaining specificity in signalling is thus far poorly understood. To address this problem we study chemosensory behaviours in C. elegans. The animal is capable of detecting at least 60 odorants and to discriminate between many of them using only two pairs of cells, AWA and AWC. Interestingly, in these cells a specific subset of 6 G subunits is expressed (Jansen ea. 1999). To study the functions of the individual G subunits we use behavioural assays including an odorant discrimination assay (Bargmann ea 1993). Our preliminary data shows that the G subunit ODR-3 is sufficient to discriminate between at least some odorants. Many downstream effectors of G-proteins are known and are being tested for their function in these pathways. However, many of these genes are expressed ubiquitously and some have severe locomotory defects. This renders behavioural assays useless to study the effects of these proteins in signalling. Therefore we are developing imaging tools to visualise responses of individual cells to olfactory or gustatory stimuli. We will use two constructs, the Yellow Cameleon (YC) (Kerr ea. 2000) and the Voltage Sensitive Fluorescent Protein (VSFP) (Sakai ea. 2001). Both constructs utilise the Fluorescence Resonance Energy Transfer (FRET) principle to measure either the calcium fluxes in the cell or changes in the membrane potential, respectively. We will drive expression using cell-specific promotors, which allows us to elucidate the signal processing on a cellular level. To study effects in olfaction we will express the constructs in AWA using the promoter of the odr-10 gene and in AWC with the gpa-13 promoter, for gustation we will use ASE (flp-5 promoter) and ASI (gpa-4 promoter). Together with the data from behavioural assays, this will provide insight in the G-protein signalling pathways in olfaction in C. elegans.

Jen-Yi Lee et al. (2005) International Worm Meeting "Wnt/Frizzled signaling regulates actomyosin contraction during C. elegans gastrulation"

Daniel Marston, Jen-Yi Lee, Bob Goldstein

[International Worm Meeting, 2005]

Cell fate specification often plays a crucial role in determining cell behaviors, such as which cells will undergo morphogenetic movements. During C. elegans gastrulation, the two endodermal precursors, Ea and Ep, move into the center of the embryo. These cells exhibit an apical/basolateral polarized localization of the PAR proteins, which is required for the accumulation of myosin (Nance et al., 2003). Furthermore, Ea and Ep undergo an apical constriction that squeezes them into the center of the embryo (Lee and Goldstein, 2003). It is unclear what other genes regulate C. elegans gastrulation. Wnt-Fz signaling has been implicated in both cell fate specification and morphogenesis in other organisms. We have evidence that mom-2/wnt and mom-5/frizzled regulate C. elegans gastrulation, in addition to their roles in cell fate specification and cell cycle timing. MOM-2 acts in a pathway independent of the polarized distribution of PAR proteins, and the absence of mom-5 does not affect myosin accumulation, suggesting that Wnt-Fz signaling may directly affect apical constriction downstream of myosin accumulation, possibly to activate contraction. In support of this hypothesis, immunostaining embryos with antibodies recognizing a phosphorylated epitope on myosin light chain (MLC) shows an enrichment of phospho-MLC at the cortex of wild-type Ea and Ep during gastrulation, whereas no such enrichment of phospho-MLC is seen in mom-5 mutant embryos. Together, these data suggest that mom-2 and mom-5 act in a signaling pathway that regulate gastrulation through activation of the actin-myosin contractile machinery.

Hannes Lans et al. (2003) International Worm Meeting "Six G subunits regulate odorant responses and olfactory neuron fate in C. elegans"

Hannes Lans, Suzanne Rademakers, Gert Jansen

[International Worm Meeting, 2003]

The olfactory system of C. elegans is ideally suited to study specificity of G-protein coupled signaling. Olfaction is mediated by two pairs of neurons, AWA and AWC. These neurons express multiple odorant receptors, enabling C. elegans to detect and discriminate between many odorants. Furthermore, six G subunits are expressed in these neurons: GPA-2, -3, -5, -6, -13 and ODR-3 (Jansen ea 1999 Nat Gen 21,414). To understand how they function, we study the different processes that are required for normal odorant responses. Here we focus on their role in odorant detection and in the establishment of AWC cell fate.We raised antibodies against all six G subunits to determine their site of action. This showed that all, except GPA-6, are localized to the ciliated endings of AWA and/or AWC, suggesting a direct role in the detection of odorants. Some were also localized to the axon. Next, we determined their role in odorant detection by testing chemotaxis of animals with loss-of-function mutations in one or more G subunits. This confirmed that ODR-3 forms the main signaling route and is sufficient to mediate olfactory perception (Roayaie ea 1998 Neuron 20,55). GPA-3 is redundant to ODR-3 and can also be sufficient for normal perception. GPA-5 is a negative regulator and GPA-2 can be both a negative and a positive regulator. GPA-13 influences signaling mostly in the context of GPA-2.The candidate odorant receptor str-2 is expressed in either the left or the right AWC neuron, indicating a functional difference between the two AWC neurons. The initial decision for this left-right asymmetry was shown to be regulated by axon contact, followed by Ca2+ and MAPK signaling (Troemel ea 1999 Cell 99,387; Sagasti ea 2001 Cell 105,221). The genes involved in these pathways include the cytoskeleton/axon guidance genes unc-44 and unc-33, the Ca2+ channel subunit unc-36, the CaM kinase II unc-43 and the MAPKKK nsy-1. We decided to study the role of the G subunits in regulating str-2 expression. For this purpose, str-2::GFP expression was determined in different G mutant backgrounds. This showed that loss-of-function of the G subunits does not affect the asymmetry of str-2 expression, but does affect its initiation and maintenance. We found that gpa-2, gpa-5, gpa-6 and odr-3 function in a pathway parallel to the Ca2+/MAPK signaling pathway formed by unc-36, unc-43 and nsy-1 to initiate str-2 expression. Similarly, gpa-5 acts in parallel to unc-44, but not unc-33. In addition, we found that gpa-6 and odr-3 may serve to maintain str-2 expression.

Jen-Yi Lee et al. (2001) International C. elegans Meeting "An in vitro System for Studying Cell Movements in Early Embryos"

Jen-Yi Lee, Bob Goldstein

[International C. elegans Meeting, 2001]

The proper migration of cells during embryogenesis is crucial for development. Gastrulation is the first major cell movement of the developing nematode C. elegans , yet surprisingly little is known about the mechanisms of this movement. We wished to use a blastomere culture system (1) to study gastrulation movements in vitro , since isolated cells can be used to address questions that cannot be addressed in intact embryos. As a first step, we asked whether or not the physical constraint of the eggshell and the underlying vitelline membrane were necessary for gastrulation. It was previously observed that gastrulation does not require the eggshell and that gastrulation-like movements may also occur in devitellinized embryos (1,2). We confirmed these observations by removing both the eggshell and the vitelline membrane and filming the embryos using a 4D microscope. Gastrulation begins with the ingression of the two E daughters, Ea and Ep, toward the center of the embryo, followed by MSpp, P 4 , MSpa, and D. We found that ingression of Ea and Ep occurs in vitro consistently (6/6 embryos), and at the time of normal gastrulation, suggesting that the in vitro system can be used to study normal gastrulation. Previous cell lysis experiments showed that an intact AB cell was not required for gastrulation (3). We wanted to further delimit the requirements for gastrulation by determining whether the progeny of an isolated, cultured P 1 cell could gastrulate. By isolating the P 1 blastomere and filming its development, we saw that gastrulation movements occurred even when only P 4 and MSpp contacted the E 2 cells. In addition, these results indicated that the normal geometry of the embryo was not essential for proper gastrulation. As with the devitellinized embryos, movements in the P 1 isolates occurred at the same time as in normal embryos. Taking advantage of the better optics resulting from reduced cell numbers, we wanted to begin to determine which cells were crawling by tracing and analyzing cell shapes in the P 1 isolates. Preliminary evidence showed that Ea and Ep remained roughly spherical, while P 4 and MSpp cells changed their shape during the time of gastrulation, making extensions towards each other, over Ea and Ep. These results suggest that the E cells do not crawl into the center of the embryo during gastrulation; rather, they appear to be pushed in by P 4 and MSpp. In vitro blastomere culture presents a useful system for studying cell migration in early development. We are currently using this system and gastrulation-defective mutants to test hypotheses relevant to how gastrulation is regulated. (1) L. Edgar, Methods in Cell Biology (1995) 48:303-321 (2) E. Schierenberg and B. Junkersdorf, Roux’s Arch Dev Biol (1992) 202:10-16 (3) J. Laufer et al., Cell (1980) 19:569-577

O'Rourke, Delia M. et al. (2013) International Worm Meeting "Does the C.elegans glycosylation gene bus-8 undergo translational frameshifting to generate different protein isoforms?"

Partridge, Frederick A, Pavlyukovskyy, Mark, Cullen, Martin, Stroud, Dave, Hodgkin, Jonathan, O'Rourke, Delia M.

[International Worm Meeting, 2013]

We have previously shown that C. elegans with missense mutations in bus-8 (a nematode-specific glycosyltransferase) are resistant to infections by M.nematophilum and Leucobacter Verde2, but hypersensitive to infection by Leucobacter Verde1. bus-8 worms are also drug-sensitive, skiddy, defective in mate recognition and bleach-sensitive due to changes in the surface coat of the worm. bus-8 is additionally essential for ventral enclosure during embryonic morphogenesis and is expressed in cells underneath the ventral epidermis in the embryo and post-embryonically in seam cells (Partridge et al. 2008). One bus-8 allele, lj22, is a point mutant in the 5'UTR that contains a conserved putative ORF out of frame with the downstream bus-8 gene. EST and RNA seq data indicate that the ORF is transcribed and spliced with the downstream bus-8 transcript. The 5'UTR ORF encodes a possible transmembrane domain that could localise the BUS-8 glycosyltransferase to membrane compartments. We hypothesize that this region is translated with the rest of the gene (which would require +1 translational frameshifting). To investigate this we constructed a TAP-tagged version of BUS-8 and transformed mutant bus-8 worms with this construct. TAP-tagged BUS-8 is functional as it can rescue both mild and severe bus-8 mutations. We have purified BUS-8 using the tags and used mass spectrometry analysis to look for peptides corresponding to the 5' UTR encoded ORF. We have also made a number of transgenic worms to investigate the functional roles of the BUS-8 isoforms. We have used genomic mutations in bus-8 that affect only the 5'UTR (lj22) or the main part of the gene (e2883, tm1410) with transgenic constructs containing modified versions of bus-8. In this way we can investigate which isoforms of BUS-8 can rescue the bus-8 phenotypes. The sensitivity of both bus-8 (lj22) and e2883 to Verde1 infection has allowed us to conduct suppressor screens. Using this approach we have identified 5 suppressors for each allele, which are currently being characterised. Partridge et al (2008) Dev Biol 317 549.

Vancoppenolle B et al. (1996) European Worm Meeting "Comparison of the early embryonic cell lineage of Caenorhabdi- tis elegans and Pellioditis marina, two rhabditid nematodes."

Vancoppenolle B, Coomans AV, Schnabel R, Borgonie G

[European Worm Meeting, 1996]

The early embryonic cell lineage of Pellioditis marina, a marine rhabditid with relatively short developing time was traced using a 4D-microscope. Although the general pattern of cell divisions is congruent with the lineage described for Caenorhabditis elegans by Sulston and coworkers, striking differences can be observed concerning migrations, timing of divisions and cell deaths. The AB, MS and C lineage of P. marina differ from those of C. elegans both in the occurence of additional cell deaths as wel as in the abscence of certain cell deaths. Additionaly, Caap does not divide in accordance with the characteristic period of the rest of the C lineage. In contrast with C. elegans, the E founder cell in P. marina undergoes a migration before gastrulation and devides into Ea and Ep only after E has entered the interior of the embryo. D and P4 divide in a similar way as in C. elegans.