Glenda Walker et al. (2001) International C. elegans Meeting "Heat Shock Factor in C. elegans and in parasitic nematodes"

Theresa Scanlon, Fiona Thompson, Andrena Brawley, Glenda Walker, Eileen Devaney

[International C. elegans Meeting, 2001]

Filarial parasites of the genus Brugia live in mammals (including humans) and are transmitted between hosts by the bite of a blood-feeding mosquito vector. The infective form for the mosquito is the L1 or microfilariae (Mf) a life cycle stage that is highly adapted for life in the bloodstream of the mammal. Mf are developmentally arrested and undergo no further development until ingested by a mosquito. The link between the progression of the developmental cycle and the transition between hosts implies that the Mf has a mechanism by which it can sense its changing environment. Results in other systems have shown that heat shock factor (HSF) can act as a cellular thermometer directly monitoring temperature and oxidative state. As temperature is one of the major differences between the mosquito and mammalian hosts we are interested in investigating the role of HSF in developmental progression. As the tools for functional genomics do not exist for Brugia, we are using C. elegans as a model system in which to define the role of HSF in a nematode. ACeDB identifies a single HSF-like gene with a highly conserved DNA binding domain. We have used an RNAi feeding vector to determine the likely function of HSF in the nematode. A number of different phenotypes were apparent at 20oC including: slow growth, scrawny appearance, thermo-sensitivity, decreased fertility and egg-laying defective. At 25oC these phenotypes were more pronounced. In an hsp-16/GFP reporter background, RNAi abolished GFP expression in the intestine but not in the pharynx or nerve ring. These results are consistent with a requirement for HSF function in the gut. Ongoing studies are focused on determining the spatial and temporal expression pattern of hsf throughout development. We aim to determine the pathways in which HSF is active under normal developmental conditions and to identify down-stream targets of HSF.

Glenda Walker et al. (2002) European Worm Meeting "Heat Shock Factor in Caenorhabditis elegans and in parasitic nematodes"

Glenda Walker, Fiona Thompson, Theresa Scanlon, Andrena Brawley, Eileen Devaney

[European Worm Meeting, 2002]

Filarial parasites of the genus Brugia live in mammals (including humans) and are transmitted between hosts by the bite of a blood-feeding mosquito vector. The infective form for the mosquito is the L1 or microfilariae (Mf) a life cycle stage that is highly adapted for life in the bloodstream of the mammal. Mf are developmentally arrested and undergo no further development until ingested by a mosquito. The link between the progression of the developmental cycle and the transition between hosts implies that the Mf has a mechanism by which it can sense its changing environment. Results in other systems have shown that heat shock factor (HSF) can act as a cellular thermometer directly monitoring temperature and oxidative state. As temperature is one of the major differences between the mosquito and mammalian hosts we are interested in investigating the role of HSF in developmental progression. As the tools for functional genomics do not exist for Brugia, we are using C. elegans as a model system in which to define the role of HSF in a nematode. ACeDB identifies a single HSF-like gene with a highly conserved DNA binding domain. Using an RNAi feeding vector containing fragments of different lengths of C.elegans HSF, we have defined a number of phenotypes. The penetrance of these phenotypes increases with the size of the RNAi fragment and higher growth temperatures. RNAi treated worms have defects in thermotolerance, lifespan, fertility and egg-laying. In addition treated worms are significantly smaller and have a scrawny appearance. In an hsp- 16/GFP reporter background, RNAi abolished GFP expression in the intestine but not in the pharynx or nerve ring. The decrease in HSP-16 levels has been confirmed by immunoblotting. Using a yolk protein/GFP reporter, flourescence in the oocytes is significantly reduced in RNAi treated worms. These results may relate to defects in gut function in RNAi treated worms, and this is consistent with a decreased gut function as defined by feeding assays. Ongoing studies are focused on determining the spatial and temporal expression pattern of hsf throughout development. Our aims are to determine the pathways in which HSF is active under normal developmental conditions and to identify down-stream targets of HSF.

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

Khan, Liakot et al. (2015) International Worm Meeting "Peri-lumenal intermediate filaments and microtubules maintain a single lumen in extending C. elegans excretory canals."

Gobel, Verena, Khan, Liakot, Membreno, Edward, Zhang, Hongjie

[International Worm Meeting, 2015]

In an RNAi-based modifier screen we identified 3 intermediate filaments (IFs: IFA-4, IFB-1, IFC-2) that enhanced the cystic ERM-1-overexpression excretory canal (EC) phenotype. Although IFB-1's function in EC morphogenesis has been noted, it remains unclear how IFs contribute to this process. Here we compare IFs' role in EC tubulogenesis with the roles of microfilaments (MFs; previously analyzed) and microtubules (MTs). IFs were found to build an intracellular perilumenal lattice, and to be non-redundantly required for lumen extension. Severe interference with IFs causes cell-body close cysts and loss of canal extension, but does not abort lumen formation. In contrast, severe interference with MFs via ERM-1/ACT-5, that we think expand the lumenal membrane via vesicle fusion, abolishes lumen formation. Selective IF removal during larval EC extension generates a "multiple lumen varicosity" phenotype with collapsed lumens between varicosities (periodic structures along extending canals that serve as osmotically sensitive "fueling stations" for wild-type growth). In contrast, larval MF removal produces short, thin lumens with small vacuoles at canal tips. IF removal generates true ectopic "lumens" (vacuoles lined with an apical membrane and cytoskeleton) not seen with MF removal. This scenario could suggest that IFs function by laterally integrating vacuoles into a lumenal membrane that has already acquired an actin coat, as opposed to MF-dependent membrane expansion via vesicle fusion at the tip that concomitantly assembles actin. A TBB-2/tubulin::GFP fusion localizes to the EC cytoplasm, but also to strands on the perilumenal IF lattice that in turn resides on perilumenal MFs. Colocalization and loss-of-function studies during EC development suggest that ERM-1 indirectly recruits lumenal IFs, whereas perilumenal lattice formation may depend on MTs. Interference with MTs copies the IF phenotypes and, unlike interference with MFs, enhances it. Thus, MTs like IFs support single lumen maintenance and lateral vacuole fusion, suggesting that one of MTs' roles in lumen extension is mediated by IFs. Effects of the 3 cytoskeletal components on endosomal and canalicular vesicles will be presented. Our findings suggest that EC lumen extension relies on tip and lateral growth and requires a resilient perilumenal IF matrix that allows vacuoles to dock at the lateral lumen (conspicuous in varicosities), thereby shaping the lumen's cylindrical tunnel structure and transmitting fluid pressure between varicosities that maintains lumen diameter and supports its anterior-posterior extension. .

Erich M Schwarz et al. (2003) International Worm Meeting "Comparative analysis of cis-regulatory sequences using four Caenorhabditis species."

Nora Mullaney, Erich M Schwarz, Tristan De Buysscher, Paul W Sternberg, Barbara J Wold, John A DeModena, Eunpyo Moon, Hiroaki Shizuya

[International Worm Meeting, 2003]

Comparing homologous cis-regulatory DNA sequences from three or more genomes has advantages over pairwise comparison of only two. Cis-regulatory sequences are short (6-20 bp) and tolerate substantial variation. Purely random pairing of unrelated 100-bp DNA segments is expected to yield two perfect 6 bp matches. Alignment of a third or fourth sequence should greatly lower the frequency of false positive regions, allowing small but real cis-regulatory sequences to be efficiently detected. This increased resolution should also allow direct comparison between phylogenetically conserved sequences and statistically overrepresented sequences, which may yield complementary views of regulatory elements. In the Caenorhabditis genus, C. remanei appears to be most closely related to C. briggsae; two other species, CB5161 and PS1010, comprise the two closest known and culturable Caenorhabditis species outside the elegans-briggsae group (Fitch, 2000). CB5161 is closest to C. elegans, and PS1010 the next most divergent; these two species thus provide an evolutionarily graded series. We have constructed fosmid libraries from CB5161 and PS1010, and begun sequencing individual fosmids for comparative analysis of genes involved in vulval or sensory neuron development. At the same time, we have devised the Mussa software package to adapt the algorithms of Davidson and coworkers (Brown et al., 2002) to multiple sequence analysis. At this writing, we have sequence data from the egl-30, lin-11, and mab-5 loci of both CB5161 and PS1010. Initial results of sequencing and comparative sequence analysis will be presented. References: Brown, C.T., Rust, A.G., Clarke, P.J., Pan, Z., Schilstra, M.J., De Buysscher, T., Griffin, G., Wold, B.J., Cameron, R.A., Davidson, E.H., and Bolouri, H. (2002). New computational approaches for analysis of cis-regulatory networks. Dev. Biol. 246, 86-102; Fitch, D.H.A. (2000). Evolution of Rhabditidae and the male tail. J. Nematol. 32, 235-244.

Michael F Portereiko et al. (2001) International C. elegans Meeting "Pharynx tubulogenesis during C. elegans development"

Michael F Portereiko, Susan E Mango

[International C. elegans Meeting, 2001]

During C. elegans embryogenesis, the pharyngeal primordium develops from a ball of cells into a linear tube connected anteriorly to the buccal cavity and posteriorly to the midgut. Using GFP reporters localized to discrete subcellular regions, we have shown that pharyngeal tubulogenesis occurs in three stages: i) lengthening of the nascent pharyngeal lumen by reorientation of apicobasal polarity of anterior pharyngeal cells (Reorientation), ii) formation of an epithelium by the buccal cavity cells, which mechanically couples the buccal cavity to the pharynx and anterior epidermis (Epithelialization), and iii) a concomitant movement of the pharynx anteriorly and the epidermis of the mouth posteriorly to bring the pharynx, buccal cavity and mouth into close apposition (Contraction) (1). We call this three-step process pharyngeal extension. We have undertaken two approaches to identify loci required for pharyngeal extension. First, we have used RNA interference to determine the role, if any, of candidate genes previously shown to be expressed in the pharynx. Second, we are undertaking a mutagenesis screen to identify mutants that generate pun (pharynx unattached) phenotypes but are otherwise largely normal. From 2000 haploid genomes, we have recovered seven Pun mutants. Our current goals are to characterize the phenotypes of the mutants and to continue screening. 1. MF Portereiko and SE Mango, Morphogenesis of the C. elegans Pharynx, Dev. Biol, in press.

Jens Schulze et al. (2007) International Worm Meeting "Cytoplasmic segregation, interphase MT and establishment of cell polarity in the embryo of the basal nematode R. culicivorax."

Einhard Schierenberg, Jens Schulze

[International Worm Meeting, 2007]

One of the central questions for developmental biologists is how cellular polarity is established and leads to blastomeres with different fate. We study nematodes from various phylogenetic positions in comparison to C. elegans in order to assess variations in the pattern of embryonic development. One of our study objects, the more basal species Romanomermis culicivorax, shows peculiarities with respect to cell polarity not observed in any other nematode. Due to differences in the orientation of cleavage spindles spatial pattern formation in R. culicivorax differs markedly from that in C. elegans. Blastomeres which in C. elegans perform a transverse, equal cleavage divide unequally with an a-p spindle orientation and vice versa. In addition, we find - so far unique among nematodes - that colored cytoplasm is segregated into a single blastomere (EMS) which inherits it to all of its descendants. Furthermore, as another phenomenon, to our knowledge not described in other animal systems, during interphase microtubule caps form in specific regions of the cortex which disappear prior to mitosis. We find these caps predominantly in cells destined to execute polar divisions indicating their involvement in cell polarisation. The EMS cell containing the brown pigment, will divide into equal left and right daughters and is the only 4-cell blastomere without interphase MT caps. Our data demonstrate that developmental variations among nematodes are more prominent and abundant than anticipated so far.

Schlosser, A. et al. (2015) International Worm Meeting "Chronic exposure to pesticide residues exacerbates alpha-synuclein pathology in a Caenorhabditis elegans Parkinson's disease model."

Flaherty, D., Schlosser, A., Davis, M.

[International Worm Meeting, 2015]

Human health issues and disease have long been linked to environmental exposures which may be the greatest risk factors for the development of neurodegenerative diseases (Franco et al. 2010). Epidemiological studies show that exposure to environmental agents such as pesticides is a key contributor to the development and exacerbation of Parkinson's Disease (PD) (Franco et al. 2010). Intraneuronal inclusions, known as Lewy bodies, composed primarily of aggregated alpha-synuclein, are the primary causative formations associated with the loss of dopaminergic neurons and the development of PD (Baltazar et al. 2014; Brown et al. 2006). The loss of these neurons causes dysfunction of the basal ganglia and the subsequent inhibition of motor control, the defining characteristic of the disease (Baltazar et al. 2014). This study looks to assess the possible toxicological effects of chronic exposure to three common pesticides, chlorpyrifos, carbaryl and indoxacarb, at maximum tolerated residue levels set by the Environmental Protection Agency (EPA) in a Caenorhabditis elegans PD model. While the EPA employs strict regulations on individual pesticides, there are no regulations governing the mixing of pesticides. In this study, pesticides are tested both individually and as binary mixtures in order to investigate alpha-synuclein toxicity. Results from the study indicate that individually, these pesticides, with the exception of indoxacarb, have a noticeable effect on alpha-synuclein pathology. When combined to form binary mixtures, all pesticides have a drastic effect on alpha-synuclein pathology and mortality, despite being within EPA limits.

Keith A Boroevich et al. (2004) West Coast Worm Meeting "Identification of Regulatory Elements in Caenorhabditis elegans using Orthology Biased Gibbs Sampling"

Steven JM Jones, David L Baillie, Keith A Boroevich

[West Coast Worm Meeting, 2004]

Much attention has recently been placed on the problem of identification of gene regulatory sequences. Two main computational approaches exist: 1) identification of sequences that share similarity to known regulatory elements, and 2) de novo identification of common motifs in a set of co-regulated genes. Orthology Biased Gibbs Sampling (OrBS) applies a modification of the Gibbs Sampler put forth by Lawrence, Altschult et al . 1 in a attempt to solve this problem. Additional features include analysis of negative strand, multiple (or no) occurrences of a motif in any given sequence and identification of multiple unique motifs, all of which have appeared in previous incarnations of the Gibbs Sampler. The unique feature of OrBS is the use of comparative genomics as an informative prior and in the calculation of motif probability. OrBS will be applied to the identification of regulatory elements C. elegans . Clustering of spatially co-regulated genes as defined by the C. elegans Gene Expression Project (see Johnsen et al . poster). This data is more amenable to regulatory element detection than the more common microarray data because the sequence in which a cis -acting element may reside is strictly defined. Interspecies sequence conservation will be identified through alignment of the C. elegans gene promoter region to the promoter region of the C. briggsae ortholog using LAGAN 2 . Predicted regulatory elements will be verified in vivo using site directed mutagenesis and promoter::GFP fusions. References: 1 Lawrence CE, Altschul SF, Boguski MS, Liu JS, Neuwald AF, Wootton JC. (1993). Science 262: 208-214. 2 Brudno M, Do C, Cooper G, Kim MF, Davydov E, Green ED, Sidow A, Batzoglou S. (2003). Genome Research 13: 721-731.

Brockett, Mary et al. (2015) International Worm Meeting "Virulence of a Novel Melanin Producing Pseudomonas Species UC17F4 on Caenorhabditis elegans."

Brockett, Mary, Morreale, Meghan, Aaronson, Lawrence, Shinn-Thomas, Jessica

[International Worm Meeting, 2015]

Pseudomonas sp. UC17F4 is a novel species of Pseudomonas isolated in our lab from the cutaneous microbial flora of the red-backed salamander, Plethodon cinereus. UC17F4 produces intracellular melanin, a brown pigment, in the presence of tyrosine-rich media. Melanin has been shown to serve as a virulence factor for several organisms, including bacteria and fungi. Our lab generated several mutant strains of UC17F4: UC17F4 (MM1) produces less melanin than the wild-type strain, UC17F4 (MM7, 8, and 9) hypersecrete extracellular melanin, and UC17F4 (PV 21 and 22) produce no melanin. We demonstrated that UC17F4 exhibits virulence by using C. elegans as a model host. Worms were transferred to lawns of UC17F4 bacteria on Nematode Growth Media (NGM) supplemented with tyrosine and lethality was assessed over time using touch assays. Our studies show that C. elegans exposed to the wild-type strain (UC17F4) have the highest mortality rate and worms exposed to UC17F4 (PV21) have the lowest mortality. Worms do not die after exposure to the hypersecreting strains or the strains without melanin. We investigated the effect of UC17F4 exposure on the different larval stages of C. elegans (L1, L2, L3, L4, and adult worms). Our results show that L1 and L2 worms are most vulnerable to the virulence of UC17F4 compared to later developmental stages. L1 and L2 worms die in less than 24 hours of exposure, whereas later developmental stages are viable and reproduce. Our current studies include determining the kinetics of L1 and L2 lethality using both touch assays and in vivo fluorescent cell death assays (SYTOX®) in a microplate reader, and determining the mode of pathogenesis using high-magnification light microscopy to examine the anatomical areas of melanin and UC17F4 biofilm accumulation. .