Marsili, J. et al. (2017) International Worm Meeting "The nematode Caenorhabditis elegans displays a chemotaxis behavior to tuberculosis-specific odorants."

Nguyen , Q.H., Voisine, C., McFall , S.M., Marsili, J., Neto, M.F., Aldakeel , S.

[International Worm Meeting, 2017]

The World Health Organization's Millennium Development Goal of halting and reversing the tuberculosis (TB) epidemic by 2015 has been met; on average, incidence of TB has fallen 1.5% per year and is now 18% lower than in 2000. Despite these gains, TB remains one of the leading causes of morbidity and mortality globally-in 2014, 9.6 million people fell ill with TB and 1.5 million died from the disease. Each year, approximately one third of the TB infected population remain undiagnosed, equating to roughly 3 million people. A simple, affordable diagnostic test for pulmonary tuberculosis (TB) is urgently needed to improve detection of active Mycobacterium tuberculosis. Recently, it has been suggested that animal behavior can be used as a biosensor to signal the presence of human disease. For example, the giant African pouched rats can detect tuberculosis by sniffing sputum specimens while honeybees respond to three of the four tuberculosis-specific volatile organic compounds (TB-VOCs) detected in the breath of TB positive patients by proboscis extension. However, both rats and honeybees require animal housing facilities and professional trainers, where few laboratories in low-income, high-burden TB settings possess the needed infrastructure. Rather than using trained animals, we propose exploiting the innate sensing capabilities of the nematode Caenorhabditis elegans, which has demonstrated reproducible, behavioral responses to over 100 VOCs. Our data shows that the innate olfactory behavioral response of C. elegans can be used to detect the TB-specific VOCs methyl p-anisate, methyl nicotinate, methyl phenylacetate and o-phenylanisole, in chemotaxis assays. It is important to note, the breath of a TB patient contains a heterogeneous mixture of TB-VOCs, although the proportionality of this blend remains unknown. To further evaluate the efficacy of C. elegans as a potential TB biosensor, we also investigated C. elegans' behavioral response to combinations of TB-VOCs, both pairwise and collectively. We found that C. elegans detects a mixture of all TB-VOCs, exhibiting a significant avoidance behavior. In addition, dauer larvae, a long-lived stress resistant alternative development state of C. elegans in which the animals can survive for extended periods of time in dry conditions with no food, were also demonstrated to detect the VOCs. Taken together, we propose a potential work-flow for a noninvasive diagnostic test for TB that incorporates collecting VOCs from the breath of the patient, exposing dauer larvae housed in a microfluidic device to the VOCs, and recording their response. These results showcase the potential for C. elegans to be incorporated into a new and affordable diagnostic test device for TB and, perhaps, other disease states, capitalizing upon its innate olfactory responses.

Samara, Chrysanthi et al. (2009) International Worm Meeting "Microfluidic in vivo screening for compounds affecting neural regeneration in Caenorhabditis elegans."

Gilleland, Cody, Norton, Stephanie, Haggarty, Stephen, Samara, Chrysanthi, Yanik, Mehmet, Rohde, Christopher

[International Worm Meeting, 2009]

Therapeutic treatment of central nervous system pathologies, such as spinal cord injuries, brain trauma, stroke, and neurodegenerative disorders, will greatly benefit from the discovery of small molecules that enhance neuronal growth after injury. Identification of a diverse repertoire of such molecules and of their cellular targets can also provide important tools for fundamental investigations of the mechanisms involved in the regeneration process. Currently, small-molecule screens for factors affecting neuronal regrowth can only be performed using simple in vitro cell culture systems. However, these systems do not truly represent in vivo environment. Importantly, off-target, toxic or lethal effects of chemical compounds can only manifest in model organisms with multiple tissue types. Thus, the thorough investigation of neuronal regeneration mechanisms requires in vivo neuronal injury models. In vivo neuronal regeneration studies have been performed mainly in mice and rats. However, their long developmental periods, complicated genetics and biology, and expensive maintenance limit large-scale studies in these animals. We previously demonstrated femtosecond laser microsurgery as a highly precise and reproducible injury method for studying axonal regeneration mechanisms in Caenorhabditis elegans1,2. Wild type nematodes move constantly, and to perform precise laser axotomy or imaging at the cellular level, animals must be immobilized. Therefore, we developed microfluidic on-chip technologies that allow automated and rapid manipulation, orientation, and non-invasive immobilization of C. elegans for sub-cellular resolution imaging and femtosecond-laser microsurgery3,4. These technologies can be used for high-throughput genetic and compound assays. We report here the results of the in vivo small-molecule screens for compounds affecting axonal regeneration after laser-induced axotomy in C. elegans. Using the technologies described above, we screened a library of small molecules. A number of compounds with a wide variety of cellular targets, such as cytoskeletal components, vesicle trafficking, and protein kinases were found to affect mechanosensory neuron regeneration following laser axotomy. REFERENCES: 1.Yanik M.F. et al., (2004). Nature. 432, 822 2.Yanik M.F. et al., (2006). IEEE J Sel Top Quant Electron. 12, 1283-91 3.Rohde C.B. et al., (2007). Proc Natl Acad Sci U S A. 104, 13891-5 4.Zeng F. et al., (2008). Lab Chip. 8, 653-6.

Sun, Lin et al. (2011) International Worm Meeting "Characterization of intracellular calcium signaling within damage C. elegans neurons ."

Gao, Fay, Roodhouse, Kevin, Sun, Lin, Chung, Samuel, Gabel, Christopher V.

[International Worm Meeting, 2011]

Traumatic neuronal damage triggers large intracellular calcium transients that are associated both with subsequent neuronal degeneration and cell death, and alternatively with regenerative repair and outgrowth [1, 2]. Combining femtosecond laser ablation with the use of genetically encoded calcium sensitive fluorophores, we can optically measure intracellular calcium signaling within a specific target neuron of an intact adult C. elegans in response to precision laser damage [3-5]. Here we characterize the damage induced calcium signal across a variety of laser ablation experiments. This includes variations in the proximity of the damage point to the cell soma, targeting of distinct morphological structures and different neuronal types, modulation of laser power, different animal ages, and multiple surgeries to the same neuron. Results are revealing complex subcellular calcium dynamics that are precisely tuned to control cell fate. We find that in general large, extended calcium transients correlate with cell degeneration, while particularly small transients are associated with reduced regenerative outgrowth. This suggests an optimum window in which elevation of cytoplasmic calcium successfully facilitates neuronal repair and outgrowth without initiating cell death. Our results are helping to pinpoint the critical aspects of this signaling pathway that dictate neuronal survival and regeneration following traumatic damage. 1.Coleman, M., Axon degeneration mechanisms: commonality amid diversity. Nat Rev Neurosci, 2005. 6(11): p. 889-98. 2.Kamber, D., H. Erez, and M.E. Spira, Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone. Exp Neurol, 2009. 219(1): p. 112-25. 3.Yanik, M.F., et al., Neurosurgery: functional regeneration after laser axotomy. Nature, 2004. 432(7019): p. 822. 4.Gabel, C.V., et al., Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans. Development, 2008. 135(6): p. 1129-36. 5.Ghosh-Roy, A., et al., Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase. Journal of Neuroscience, 2010. 30(9): p. 3175-3183.

James D McGhee et al. (2005) International Worm Meeting "Efficient specification of the C. elegans endoderm does not require zygotic expression of the med-1 and med-2 genes."

Barbara Goszczynski, James D McGhee

[International Worm Meeting, 2005]

The med-1 and med-2 genes encode a pair of essentially identical GATA factor- related transcription factors that have been proposed to be necessary for specification of the entire C. elegans endoderm (intestine or E lineage) as well as MS-derived mesoderm [1]. med-1 and med-2 are proposed to be the direct downstream targets and the principal effectors of the maternally provided SKN-1 transcription factor; med-1 and med-2 would thus occupy the pivotal interface between maternal and zygotic control of gene expression. The conclusion that med-1 and med-2 are essential for C. elegans endoderm specification was based on a partially-penetrant (~50%) loss of endoderm markers produced by RNA-mediated interference (RNAi) [1]. To determine whether this partial penetrance reflects: (i) inefficient RNAi against early zygotic transcripts; (ii) approximately the expected level of endoderm loss in skn-1 nulls, or; (iii) a further redundancy in the pathway of endoderm specification, we constructed worm strains that segregate embryos lacking both the med-1 gene (because of a gene-specific deletion) and the med-2 gene (using either of two overlapping chromosomal deficiencies). Contrary to expectations, we observe that the large majority (80 - 97 percent) of med-2(-); med-1(-) embryos still express markers of endoderm differentiation. Thus, we conclude that zygotic expression of the med-1 and med-2 genes are not necessary to specify the C. elegans endoderm. Our results could possibly suggest that the C. elegans endoderm can be efficiently specified by a new pathway involving a maternal contribution from the med genes and that (presumably) does not require SKN-1. However, any maternal contribution of the med genes has so far resisted conventional RNAi (maternal injection), under conditions where parallel injections with control dsRNAs (e.g. glp-1, let-413) are highly effective. 1.Maduro, M.F., Meneghini, M.D., Bowerman, B., Broitman-Maduro, G., and Rothman, J.H. (2001). Restriction of mesendoderm to a single blastomere by the combined action of SKN-1 and a GSK-3beta homolog is mediated by MED-1 and -2 in C. elegans. Mol Cell 7, 475-485.

Shaw, W. Robert et al. (2009) International Worm Meeting "The miR-51 Family Of microRNAs Is Required For The Maintenance Of Pharyngeal Attachment."

Shaw, W. Robert, Miska, Eric A.

[International Worm Meeting, 2009]

A deletion screen (1) has developed a resource of mutants carrying deletions in most C. elegans microRNAs (miRNAs), short regulatory RNAs that act to repress gene expression post-transcriptionally. The function of most miRNAs in C. elegans is unknown and the majority of mutants show no gross abnormal phenotype (1). The miR-51 family is a redundant and conserved family of six miRNAs whose putative promoters drive GFP in overlapping but not identical spatial patterns. Mutants lacking multiple members of the miR-51 family show gradually more severe defects in growth and animals lacking all members of the family arrest as larvae with an unattached pharynx (Pun) phenotype. These mutant phenotypes can be rescued by short genomic regions encoding any of the miR-51 family miRNAs, demonstrating their redundancy. The pharynx of C. elegans forms from a ball of cells specified in the interior of the developing embryo and extends anteriorly to attach to the hypodermis (2). This attachment requires the arcade cells, which become polarised during pharyngeal attachment and form a continuous epithelium between the pharyngeal and hypodermal cells (2). In the early development of mir-51 family mutant larvae, this epithelium is labelled with a DLG-1-mCHERRY fusion protein indicating that the arcade cells have polarised. However, the attachment of the cells to the pharynx is not maintained and arcade cells separate from the anterior pharynx. We have identified the first target mRNA of the miR-51 family of microRNAs as the cadherin, cdh-3. A reporter for cdh-3 drives GFP expression in the arcade cells and the 3''UTR of cdh-3 mRNA confers direct regulation by the miR-51 family of miRNAs. However, mutations in cdh-3 or cdh-3(RNAi) fail to suppress the Pun phenotype, suggesting the regulation of a network of miR-51 family target mRNAs is important in the maintenance of pharyngeal attachment. (1) Miska, E. A., Alvarez-Saavedra, E. A., Abbott, A. L., Lau, N. P., Hellman, A. B., McGonagle, S., Bartel, D. P., Ambros, V. R., Horvitz, H. R. - PLoS Genetics, 2007) (2) Portereiko, M.F., Mango, S.E. - Developmental Biology (2001).

Yueyi I Liu et al. (2004) West Coast Worm Meeting "Global Identification of C. elegans Regulatory Motifs"

Stuart K Kim, Yueyi I Liu

[West Coast Worm Meeting, 2004]

Regulatory motifs are short sequences of DNA that regulate the level, timing, and location of gene expression. Identifying these motifs and their functions is crucial in our understanding of gene regulation and disease processes. We developed CompareProspector, a motif-finding program that takes advantage of cross-species sequence comparison to identify putative regulatory motifs from sets of co-regulated genes [1] . We applied CompareProspector to 30 sets of genes with very similar patterns of expression, identified from the C. elegans topomap [2] and individual DNA microarray experiments. The statistical significance of each candidate motif identified was evaluated using criteria such as motif enrichment-the ratio of prevalence of the motif in a given set of promoters to its prevalence elsewhere in the genome, and the expression coherence of genes with the motif. We identified twelve significant regulatory motifs, three of which have literature evidence confirming they are true regulatory motifs. Overall, these twelve motifs are found in the upstream regulatory regions of 2970 different genes, and may be involved in gene regulation in 24 clusters of co-expressed genes. The first known motif, with the consensus TGATAA, matches the consensus of known binding sites for GATA factors. As GATA factors are known to be involved in worm intestine development [3] and hyperdermis development, it is not surprising that the GATA motif is identified from a set intestine-specific genes (F. Pauli, unpublished), mount08 of the topomap, which is enriched in genes from the intestine, and several collagen-related datasets (mount14, 17, and 35 of the topomap). We correctly identified GATA sites in the promoters of genes known to be regulatory by GATA factors. Interestingly, the GATA motif is also identified from several data sets involved in the aging process. This result parallels that of Murphy and colleagues, who independently identified this motif from their data set of DAF-16 target genes [4] . Both our result and the result from Murphy suggest that GATA factors may be involved in worm aging. Motif 2, which is identified in the two heat shock-related data sets, matches the consensus of known binding sites for heat shock factors [5] . Motif 3 matches the consensus of heat shock associated sites (HSAS), a motif that was first predicted computationally to be involved in the heat shock process [6] and later experimentally validated to be involved in ethanol stress response (14 th International C. elegans Conference abstract 1113C). We are currently in the process of validating the rest of the motifs and their individual binding sites using mutagenesis studies of promoters with predicted motifs. 1. Liu, Y., Liu, X.S., Wei, L., Altman, R.B. and Batzoglou, S. (2004) Eukaryotic regulatory element conservation analysis and identification using comparative genomics . Genome Res. 14 , 451-8. 2. Kim, S.K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart, J.M., Eizinger, A., Wylie, B.N. and Davidson, G.S. (2001) A gene expression map for Caenorhabditis elegans . Science. 293 , 2087-92. 3. Maduro, M.F. and Rothman, J.H. (2002) Making worm guts: the gene regulatory network of the Caenorhabditis elegans endoderm . Dev Biol. 246 , 68-85. 4. Murphy, C.T., McCarroll, S.A., Bargmann, C.I., Fraser, A., Kamath, R.S., Ahringer, J., Li, H. and Kenyon, C. (2003) Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans . Nature. 424 , 277-83. 5. Amin, J., Ananthan, J. and Voellmy, R. (1988) Key features of heat shock regulatory elements . Mol Cell Biol. 8 , 3761-9. 6. GuhaThakurta, D., Palomar, L., Stormo, G.D., Tedesco, P., Johnson, T.E., Walker, D.W., Lithgow, G., Kim, S. and Link, C.D. (2002) Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods . Genome Res. 12 , 701-12 .