Hunter, Skyler et al. (2015) International Worm Meeting "Caenorhabditis Sieve: A novel mechanical approach to sorting C. elegans and other nematode species for age-synchronized, large scale assays."

Hunter, Skyler, Scherbak, Courtney, Taylor, Barbara, Vayndorf, Elena

[International Worm Meeting, 2015]

The most common method of C. elegans transfer is to use a fine sterilized "pick" made of hair or wire to move animals individually or in small groups. Many procedures, such as RNA extraction or fluorescent imaging, require a large sample of synchronized animals of reproductive age for accurate results. Without expensive sorting equipment, researchers are left to manually select and transfer individual animals. To address this need, we built a low-cost (under $10), easy-to-make device to efficiently select and move large numbers of nematodes from plate to plate without the use of a pick, FUDR, or high-cost sorting machines. Our transfer procedure uses a filtration device created with common lab supplies, and a standard buffer solution. The conceptual approach is to filter a population of animals through a fine mesh attached to a plastic cap, then rinse off leftover unwanted animals and debris from the mesh into a disposable tube. The device is then flipped over and the selected animals are rinsed off the filter into a collection tube where they can settle by gravity, after which they are pipetted into or onto a fresh media source. We will present data comparing our method to the current pick selection method in terms of percent yield of filtered animals, use in large scale assays, and several health parameters, including motility, pump rate, fecundity, and activation of stress response genes.

Weiner A (2003) Mol Cell "Soaking up RNAi."

Weiner A

[Mol Cell, 2003]

RNA interference (RNAi) describes the ability of double-stranded RNA (dsRNA) to inhibit homologous gene expression at the RNA or DNA level. In a recent paper, Feinberg and Hunter report that a single transmembrane dsRNA transport protein may enable RNAi to spread systemically from one cell to another.

Maulik, Malabika et al. (2017) International Worm Meeting "Alaskan Blueberry attenuates alpha-synuclein over expression and improves movement in Parkinson's like model of Caenorhabditis elegans."

Mitra, Swarup, Maulik, Malabika, Taylor, Barbara, Vayndorf, Elena, Hunter, Skyler, Bult-Ito, Abel

[International Worm Meeting, 2017]

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons and aggregation of alpha-synuclein (AS) protein leading to motor and cognitive impairment. The current study investigates the role of Alaskan bog blueberry (Vaccinum uliginosum), on alpha synuclein aggregation using a transgenic model of Caenorhabditis elegans expressing human alpha-synuclein [OW13 (P(unc-54)::alpha-synuclein::YFP+unc-119)].The current study also examines the role of Sirtuin 1, a histone deacetylase, in reducing the toxicity of alpha-synuclein aggregates and whether this effect is mediated via expression of other downstream molecular targets. The Alaskan bog blueberry was chosen for its high phenolic content, because phenolics have been shown to modulate sirtuin-mediated molecular pathways. Our experiments showed that the crude extract of low bog blueberry ( 100 and 400 ug/ml) reduced alpha-synuclein aggregation and improved motility in the worm model. The study also highlights the molecular mechanism through which the botanicals are exerting this beneficial effect. These findings encourage further studies on these Alaskan botanicals as possible therapeutic agents for Parkinson's disease, specifically of interest are the identification of active ingredients within the extracts and their optimal doses.

Michael C Fitzgerald et al. (2004) East Coast Worm Meeting "Specificity of the C. elegans Putative Transmembrane Channel SID-1"

Craig P Hunter, Michael C Fitzgerald

[East Coast Worm Meeting, 2004]

SID-1 was identified in a genetic screen for mutants capable of cell autonomous RNAi but deficient for systemic RNAi (Winston et al., 2002). The systemic RNAi defect is likely due to the inability of cells lacking SID-1 to import double-stranded RNA (dsRNA) from neighboring cells. The nature of SID-1 activity was elucidated using a heterologous system whereby either wild-type SID-1 or, as a negative control, a missense mutant form of SID-1 was transiently expressed in Drosophila S2 cells (Feinberg & Hunter 2003). These investigations showed that SID-1 enables efficient RNAi by adding dsRNA to the media of transfected S2 cells (soaking RNAi) and uptake of labeled dsRNA into cells. Furthermore, long dsRNA was shown to be more effective than short dsRNA for SID-1 mediated soaking RNAi in S2 cells and for systemic RNAi in C. elegans . We are investigating the length dependence of silencing as well as substrate specificity of the SID-1 channel. We have shown that SID-1 is extremely efficient, enabling soaking RNAi with less than one molecule of dsRNA per transfected cell and will present evidence that transport is extremely rapid. We will also report the results of ongoing analyses of length-dependent transport and channel selectivity, which may have implications for the use of SID-1 as a tool for molecular biology. W. M. Winston, C. Molodowitch, C. P. Hunter, Science 295 , 2456-59 (2002). Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. E. H. Feinberg, C. P. Hunter, Science 301 , 1545-7 (2003). Transport of dsRNA into cells by the transmembrane protein SID-1.

Julia M Claggett et al. (2004) East Coast Worm Meeting "An Investigation into the potential regulatory relationships between components of a network specified by pal-1."

L Ryan Baugh, Julia M Claggett, Craig P Hunter

[East Coast Worm Meeting, 2004]

PAL-1 protein, contributed both maternally and zygotically, is necessary and sufficient to specify and maintain the C blastomere lineage in the C. elegans embryo (Hunter and Kenyon, 1996). A number of this master regulator's targets were identified by microarrays comparing the transcript abundance in wild-type and mutant embryos either lacking or containing extra C blastomeres. Furthermore, we collected these embryos at defined time points, thus additionally providing temporal information. Target genes could then be separated by their transcriptional initiation into four consecutive temporal phases defined by a singular cell cycle beginning with the 2C-cell stage (Baugh et al, 2003). Using reporter YFP constructs for thirteen of the targets and a volume-rendering program, the 3D spatial expression pattern of each target gene was established. On the basis of this spatial information and knowledge of the temporal phase to which each target belongs, we have proposed a set of regulatory relationships between the components. We are currently testing these hypotheses by disrupting potential (capital O, grave accent)upstream(capital O, acute accent) regulators via RNAi and/or mutation and either observing the effect on individual (capital O, grave accent)downstream(capital O, acute accent) reporters or analyzing the effect on transcript abundance using QPCR. We hope that such measurements will give us insight into how the genes within the pal-1 network regulate each other in order to establish and maintain the various cell fates within the C blastomere lineage. Hunter, C.P. and Kenyon, C. (1996). Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos. Cell 87, 217-26. Baugh, L.R., Hill, A.A., Slonim, D.K., Brown, E.L. and Hunter, C.P. (2003). Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome. Development 130, 889-900.

Vayndorf, Elena et al. (2015) International Worm Meeting "Protein homeostasis dysregulation is associated with aberrant morphology and reduced function of aging mechanosensory neurons."

Hunter, Skyler, Driscoll, Monica, Toth, Marton, Scerbak, Courtney, Taylor, Barbara, Neri, Christian, Parker, J. Alex, Vayndorf, Elena

[International Worm Meeting, 2015]

In both C. elegans and humans, the aging nervous system is characterized by decreased synaptic activity, deteriorating short-term and long-term memory, and altered neuronal morphology. Given the overwhelming evidence for proteostasis disruption in neuronal aging, we sought to explain the accumulation of neuronal morphological abnormalities by focusing on protein homeostasis in 6 mechanosensory neurons of aging C. elegans nematodes. We examined the effects of disrupted proteostasis on the integrity of neuronal cytoarchitecture using a transgenic model with an excessively high neuronal protein load, and RNAi knock down of specific genes involved in protein turnover. We found that animals expressing the first 57 amino acids of the human huntingtin gene and an expanded polyglutamine CAG tract (Q128) in mechanosensory neurons accumulate more aberrations that are distinct from those found in animals that express the non-toxic (Q19) number of repeats, or those that express no repeats. We scored and tallied these changes in both the soma and processes and found that they are sometimes associated with improved or reduced function. Next, we used an RNAi candidate gene approach to target genes involved in the maintenance of protein homeostasis in wild-type animals. We found that genes involved in protein turnover play an important role in maintaining the integrity of healthy neurons, and that their knockdown leads to distinct morphological changes in both the process and the soma of wild-type mechanosensory neurons. Taken together, these results suggest that protein homeostasis is critical for maintaining neuronal integrity and function, and that disrupted proteostasis contributes to morphological abnormalities that occur more frequently with advanced age.

Vayndorf, Elena et al. (2013) International Worm Meeting "Protein homeostasis dysregulation drives aberrant morphology of aging mechanosensory neurons."

Neri, Christian, Parker, J. Alex, Toth, Marton, Nichols, Courtney, Vayndorf, Elena, Taylor, Barbara, Driscoll, Monica, Hunter, Skyler, Parker, Cyrena

[International Worm Meeting, 2013]

In both C. elegans and mammals, the aging nervous system is characterized by decreased synaptic activity, deteriorating short-term and long-term memory, and altered neuronal morphology. We sought to elucidate the functional consequences of altered neuronal morphology by focusing on protein homeostasis in individual neurons. We examined the effects of disrupting proteostasis on the integrity of neuronal cytoarchitecture using a transgenic model with an excessively high neuronal protein load. We found that animals expressing the first 57 amino acids of the human huntingtin gene and an expanded polyglutamine CAG tract (128Q) in mechanosensory neurons accumulate significantly more neuronal aberrations and more protein aggregates, and have a significantly greater decline in function with age, compared to animals that express the non-toxic (19Q) number of repeats, or those that express no repeats. We identified specific morphological alterations, in particular extreme outgrowths in the soma of ALM mechanosensory neurons, as well as a wavy phenotype in processes of PLM neurons, as the major aberrant morphological types in this transgenic background. Our RNAi studies suggest that targeting genes expressed in organelles associated with the maintenance of proteostasis, in particular the proteosome, lysosome and endoplasmic reticulum, alters neuronal morphology and accelerates aging in wild-type animals. Taken together, these results suggest that protein homeostasis is critical for maintaining neuronal integrity and that disrupted proteostasis contributes to morphological abnormalities that increase in frequency with age.

Tawe WN et al. (1997) Worm Breeder's Gazette "Paraquat mediates differential gene expression in C. elegans"

Henkle-Duehrsen K, Tawe WN, Eschbach M-L, Walter RD

[Worm Breeder's Gazette, 1997]

In order to identify genes that are differentially expressed as a consequence of stress due to paraquat, we used the differential display technique (1) to compare mRNA expression patterns in Caenorhabditis elegans. A C. elegans mixed-stage worm population, as well as a homogeneous larval population were treated with 100 mM paraquat, parallel to controls. Over 50 mRNA species that are potentially up-regulated in response to paraquat were identified. Sixteen of these candidates were re-amplified, ligated into plasmid vectors and the nucleotide sequences were determined. The induction of four of these expressed sequence tags (ESTs) designated L1, M47, M96 and M132 were confirmed in two independent stress / differential display experiments, as well as by northern blot analysis with RNA from stressed and unstressed worms. The nucleotide sequences of the independently isolated L1 and M47 ESTs were found to be identical. Their corresponding mRNA level increased more than 40-fold in the larval stage, and to a lesser extent in the mixed-stage worm population, in response to paraquat. Induction levels of 3 - 5 fold were observed for the M132 and M96 ESTs, in both larval and mixed populations. All of the isolated ESTs showed homology to portions of the C. elegans cosmids. The L1/M47 EST is derived from a gene encoding one of the putative C. elegans glutathione S-transferases. The paraquat-inducible M132 EST was also identified, and encodes a putative C2H2 - type zinc finger protein, possessing an N-terminal leucine zipper. However, the M96 EST appears to be a novel gene whose product has not yet been identified. Searches of other eukaryotic nucleotide sequence databases did not reveal any significant homologies to known sequences from any organism. Since paraquat is known to generate superoxide radicals in vivo, and the cellular superoxide dismutase (SOD) enzyme complex is responsible for the quenching of the deleterious effects of these radicals, the response of the C. elegans superoxide dismutases (2,3,4) to paraquat was also investigated in this study. Northern blot experiments demonstrated that mRNA steady state levels of the C. elegans manganese type and the copper/zinc type superoxide dismutases increased two-fold in response to paraquat, in the larval population. In contrast, mixed-stage populations did not show any apparent increase in the levels of these SOD mRNAs in response to paraquat. References: 1. Liang, P., Pardee, A.B. (1992). Science 257: 967 - 971 2. Giglio, M-P., Hunter, T., Bannister, J.V., Bannister, W.H., Hunter, G.J. (1994). Biochem. Mol. Biol. Int. 33 (1): 37-40 3. Giglio, A. M., Hunter, T., Bannister, J.V., Bannister, W.H., Hunter, G.J. (1994) . Biochem. Mol. Biol. Int. 33 (1): 37-40 4. Larsen, P.L. (1993). Proc. Natl. Acad. Sci. USA, 90: 8905-8909

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).

Joanne C Wen et al. (2003) International Worm Meeting "Functional Characterization of Early Embryonic PAL-1 Targets"

Craig P Hunter, Joanne C Wen, L Ryan Baugh

[International Worm Meeting, 2003]

The activation and maintenance of C lineage specification occurs through maternal and zygotic PAL-1 activity, respectively (Hunter & Kenyon, 1996; Edgar et al, 2001). A set of targets of this master regulatory transcription factor were identified by transcript profiling embryos with perturbed PAL-1 activity (see abstract by Baugh et al). To functionally characterize PAL-1 targets, we have used RNAi to assess the lethality and terminal phenotypes following loss of function. To identify interactions between targets, we are performing epistasis analysis both by scoring synthetic lethality and by examining the effect of RNAi against one target on the expression of reporters for other targets. Our hope is that such functional characterization of a key set of PAL-1 targets will generate the data necessary to begin modeling the PAL-1 regulatory network.