Chavez, Ivan et al. (2021) International Worm Meeting "Skin-penetrating nematodes exhibit life-stage-specific interactions with host-associated and environmental bacteria"

Chavez, Ivan, Bryant, Astra, Assie, Adrien, Samuel, Buck, Hallem, Elissa, Brown, Taylor

[International Worm Meeting, 2021]

Skin-penetrating nematodes of the genus Strongyloides infect over 600 million people, posing a major global health burden. Their life cycle includes both a parasitic and free-living generation. During the parasitic generation, infective third-stage larvae (iL3s) actively engage in host seeking. During the free-living generation, the nematodes develop and reproduce on host feces. At different points of their life cycle, Strongyloides species encounter bacteria from various ecological niches. However, the microbial interactions between Strongyloides and bacteria remain uncharacterized. We first investigated the microbiome of the human parasite Strongyloides stercoralis using 16S-based amplicon sequencing. We found that S. stercoralis free-living adults have a distinct microbiome, suggesting that they selectively associate with specific fecal bacteria. We then investigated the behavioral responses of S. stercoralis and the closely related rat parasite Strongyloides ratti to an ecologically diverse panel of bacteria. We found that S. stercoralis and S. ratti showed similar responses to bacteria. The responses of both nematodes to bacteria varied dramatically across life stages: free-living adults were strongly attracted to most of the bacteria tested, while iL3s were attracted specifically to soil bacteria. The behavioral responses to bacteria were dynamic, consisting of distinct short- and long-term behaviors. Finally, a comparison of the growth and reproduction of S. stercoralis free-living adults on different bacteria revealed that the bacterium Proteus mirabilis inhibits S. stercoralis egg hatching, greatly decreasing parasite viability. Our results identify bacteria that serve as key sensory cues for directing movement, as well as bacteria that decrease the parasite's reproductive fitness.

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).

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.

Matai, Latika et al. (2015) International Worm Meeting "Oxidative quality control regulates response to ER stress through translation control."

Matai, Latika, Rajkumar, Asher, Sengupta, Shantanu, Maity, Shuvadeep, Mukhpadhyay, Arnab, Chakraborty, Kausik

[International Worm Meeting, 2015]

The Unfolded protein response is a signalling network that is triggered by the accumulation of misfolded proteins within the ER lumen, a condition termed as ER stress. Importantly, with progressing age, the ability of an organism to mount an effective response to ER stress declines significantly (Taylor and Dillin, 2013), the reason if resolved completely could have tremendous implications in aging research. In a comprehensive genetic screen to identify modulators of reductive stress-induced UPRER in S. cerevisiae, we found that oxidative quality control (OQC) genes modulate the cellular response to chronic reductive stress. Further studies in Caenorhabditis elegans revealed that ROS accumulation through pharmacological or genetic interventions results in non-canonical translation attenuation, blocking UPRER. Interestingly we find ROS accrual to be a potent reason for age related decline in iUPRER. We also show evidence that, ironically, the evolution of Perk-dependent translation attenuation system allows higher eukaryotes to bypass ROS-dependent non-canonical mode of translation attenuation by decreasing protein load in the ER.Keywords: ROS: Reactive oxygen species, iUPRER: induced UPRER.

Barrett, Alec et al. (2021) International Worm Meeting "Integrating bulk and single cell transcriptomics for accurate detection of tissue-specific gene expression"

Barrett, Alec, Taylor, Seth R., Weinreb, Alexis, Miller III, David M., Varol, Erdem, Sestan, Nenad, Hobert, Oliver, McWhirter, Rebecca, Li, Mingfeng, Hammarlund, Marc

[International Worm Meeting, 2021]

Advances in RNA-seq for bulk and single cell (sc) approaches have produced increasingly fine dissections of the C. elegans transcriptome. Although both techniques can yield transcriptomes for individual cell types, each comes with strengths and weaknesses. scRNA-Seq affords high resolution, but suffers from dropout, leading to false negatives. Bulk sequencing detects more genes, but suffers from contaminating cell types, resulting in false positives. In this work we integrated these orthogonal approaches to improve the accuracy of both methods. We used bulk samples collected for specific neuron types and sc datasets for all C. elegans neurons and additional non-neuronal cells (1). We used sc data to estimate contamination in each bulk sample, and developed novel methods for removing these gene counts. In one approach we used linear histogram matching to scale sc counts, and subtracted putative contamination using data from non-neuronal clusters. In another approach we used bootstrapping to estimate gene level contributions from target and contaminating tissues in sc data and apply them to bulk counts, providing a bootstrap sample distribution of corrected expression data. We assessed these approaches in two ways: 1) Measuring improvements in calling genes with known expression in all neurons; 2) Examining effects on eliminating genes expressed exclusively in contaminating tissues. We found that our analysis reduced false positives, while maintaining robust true positive detection, thus offering a unique strategy for utilizing complementary bulk and sc RNA-Seq data sets to enhance the accuracy of cell-specific expression profiling data. 1. Taylor SR, Santpere G, Weinreb A, Barrett A, Reilly MB, Xu C, et al. Molecular topography of an entire nervous system. bioRxiv. 2020:2020.12.15.422897.

Ooi, Felicia et al. (2015) International Worm Meeting "Uncovering the mechanisms by which the neurosensory release of serotonin modulates the Heat Shock Response in Caenorhabditis elegans."

Ooi, Felicia, Prahlad, Veena

[International Worm Meeting, 2015]

Recent advances in the field have established that the Heat Shock Response (HSR) in the nematode Caenorhabditis elegans can be controlled in a cell non-autonomous manner (Prahlad et al, 2008; Prahlad & Morimoto, 2011; Maman et al, 2013; Taylor & Dillin, 2013; van Oosten-Hawle et al, 2013; Kumsta et al, 2014). One mechanism of such cell non-autonomous control is through AFD thermosensory neuronal stimulation of serotonin (5-HT) release from the ADF serotonergic neurons (Tatum et al, 2015). Of note, optogenetic stimulation of 5-HT release can activate HSF-1 and transiently upregulate hsp70 mRNA, even in the absence of actual heat stress or protein misfolding. We have identified SER-1, a 5-HT2-like metabotropic G-protein coupled receptor, as having a role in the HSR signaling pathway downstream of 5-HT release, as ser-1 mutants demonstrate a diminished HSR when subjected to either heat shock or optogenetic stimulation of serotonergic neurons. In contrast to actual heat shock, the excitation-coupled release of 5-HT is sufficient to suppress polyglutamine aggregation in C. elegans muscle cells. These studies establish that neurosensory perception of stress is linked to repair of cellular damage. In addition they suggest that the regulation of HSF-1 by serotonergic signaling may differ from that which occurs during heat shock since the commonly accepted model of HSF-1 activation upon heat shock is thought to be through the titration of chaperones by protein misfolding. We are currently using genetics and RNAi screens to investigate whether and how HSF-1 activation by serotonergic signaling is distinct from that induced upon heat shock.

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.

J Kass et al. (1999) International C. elegans Meeting "The EGL-3 prohormone convertase modulates ASH-interneuron synapses"

JM Kaplan, J Kass, P Kim

[International C. elegans Meeting, 1999]

How do animals distinguish between sensory stimuli? We are studying a neural circuit in C. elegans in which the ASH sensory neuron, detect noxious chemical and mechanical stimuli and signal to the worm to move backward. Three stimuli are detected by the ASH neurons: nose touch, high osmolarity and volatile repellants. Genetic and molecular evidence suggests that different signals are produced at the ASH-interneuron synapses in response to the different stimuli. GLR-1 glutamate receptors are expressed in interneurons, are clustered at ASH-interneuron synapses, but are required specifically ASH-mediated touch sensitivity. We isolated egl-3(nu349) as a suppressor of glr-1(n2461) touch insensitivity, Alleles of egl-3 cause three phenotypes: they are egg-laying defective, insensitive to gentle touch to the body, and they suppresses the nose touch defect of glr-1 . The egl-3 mutation restores function of the ASH-interneuron synapses because glr-1(n2461); egl-3(nu349) double mutants became nose touch defective after ablation of ASH. The egl-3 mutants were also defective for habituation of the nose touch response. We have cloned egl-3 , and found that it encodes a homolog of Prohormone Convertase 2, a neuroendocrine-specific protease (C51E3.7). Two egl-3 alleles correspond to mutations in predicted exons of C51E3.7: n150ts contains two missense mutations, in the catalytic domain and the homoB domain; nu349 contains a missense mutation in the homoB domain. The homoB domain has been shown to be required for maturation and secretion of the protease (Taylor et al., 1998). Both alleles are recessive. These results suggest that a neuropeptide (processed by EGL-3) inhibits ASH-interneuron signaling, and is involved in habituation to repeated sensory stimuli.

Hall, David H. et al. (2009) International Worm Meeting "Electron tomography: a new tool for exploring C. elegans cellular anatomy."

Fisher, Kevin, Nguyen, Ken C.Q., Hall, David H., Crocker, Chris, Derr, KD, Rice, William J., Politi, Kristin A., Gunther, Leslie

[International Worm Meeting, 2009]

C. elegans has been studied intensively using serial section reconstruction for several decades, so that every cell type is now known in considerable detail. Nevertheless, limitations of the serial section technique (notably the poor resolution of detail within the depth of each 50 nm "thin" section) have made it difficult to model the shapes of fine details in many organelles, such as the cristae of a mitochondrion, the canaliculi of the excretory canal, membrane ruffles in many cell types, or the constituents at a chemical synapse. High pressure freeze fixation and freeze substitution (1) are a required element in preserving smaller structures that generally escaped our notice in conventional TEM imaging. Modern electron microscopes using higher energy electrons now offer much better resolution by collecting multiple images in a tilting series through comparatively thick sections (150 nm) using the SerialEM program (2). The Protomo software package (3) is used to compute a 3D tomographic reconstruction that offers the same level of detail in any dimension (roughly 2 nm resolution). Annotation and modeling is done using IMOD (4) and/or Amira. This permits us to take a new look at many familiar objects in the anatomy of C. elegans, to identify missing parts of the whole anatomy, and potentially, to detect smaller anatomical defects in a variety of mutant backgrounds. Here we will introduce the tomographic procedure, and share finished 3D models of some typical intracellular organelles at high resolution. Supported by NIH RR 12596 to DHH. 1.Weimer, R.M. (2006) Methods Mol. Biol. 351: 203-221. 2.Mastronarde, D.N. (2005) J. Struct. Biol. 152: 36-51 3.Winkler, H. and Taylor, K.A. (2006) Ultramicroscopy 106: 240-254. 4.Kremer, J.R., Mastronarde, D.N. and McIntosh, J.R. (1996) J. Struct. Biol. 116: 71-76.

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.