Amigoni, L. et al. (2019) International Worm Meeting "Caenorhabditis elegans as a model for searching compounds capable of preventing Spinocerebellar Ataxia Type 3."

Bonanomi, M., Visentin, C., Sciandrone, B., Airoldi, C., Amigoni, L., Tortora, P., Regonesi, M.E., Natalello, A.

[International Worm Meeting, 2019]

Ataxin-3 (ATX3) is the protein that triggers the neurodegenerative disorder spinocerebellar ataxia type 3 (SCA3) when its polyglutamine stretch exceeds the critical length of 55 residues. This expansion results in misfolding and structural rearrangements, leading to aberrant interactions and the consequent formation of fibrillar amyloid-like aggregates. Currently, no effective treatment is available for SCA3 disease. In order to find natural compounds able to prevent ATX3 aggregation, we studied the effect of tetracycline (TET) and epigallocatechin-3-gallate (EGCG) on ATX3 fibrillogenesis. We displayed that TET increased the solubility of the different aggregated species, without affecting the secondary structures of the final aggregates. Otherwise, EGCG interfered with the early steps of aggregation, accelerating the Josephin Domain (JD) misfolding, and leading to the formation of off-pathway, non-amyloid, final aggregates. By NMR analyses, we proved that the different behavior could be related to the capability of the sole EGCG to bind the monomeric JD. Successively, among a small library of tetracycline, we identified methacycline (MET) as the most effective compound, which exerts the same mechanism of action of TET. The pharmacological efficacy of EGCG, TET and MET has been evaluated on a SCA3 C. elegans model and we demonstrated that all compounds induced a significant increase in mobility without extending the lifespan. MET was proven to be the most effective in relieving the locomotion defects. Even more remarkably, MET treatment also resulted in an ameliorated motility even in wild-type worms suggesting that MET fulfills its action via diverse mechanisms, besides interfering with amyloid aggregation. Furthermore, the drug did not significantly modify the life span in any of the strains investigated, which supports the expectation that it may be devoid of toxic effects in long-term administration.

JD Kormish et al. (1999) International C. elegans Meeting "A genetic screen for genes involved in gut development and differentiation"

JD McGhee, JD Kormish

[International C. elegans Meeting, 1999]

To investigate genes potentially involved in gut development and differentiation, we are developing a genetic screen for gut obstructed (gob) mutants. The gut specific elt-2 GATA factor appears necessary for correct gut cell development. elt-2 null mutants arrest as L1s with a blocked intestinal lumen and abnormal brush border (1). When elt-2 null mutants are fed on a mixture of fluorescent beads and Escherichia coli , the beads collect as a large "gob" in the posterior pharynx and anterior gut that can be easily detected under the dissecting microscope. We are now using this gut obstructed phenotype to screen for mutants that should potentially identify genes involved in gut development. Preliminary results of this screen are promising, as several candidates have already been isolated. 1) Fukushige T, Hawkins MG and McGhee JD (1998) Dev Biol 198(2):286-302

Meisel, Joshua et al. (2015) International Worm Meeting "Genetic Analysis of Neuronal Signaling Coupling Microbial Detection to the Induction of TGF-beta/DAF-7 Gene Expression in C. elegans."

Kim, Dennis, Meisel, Joshua

[International Worm Meeting, 2015]

Environmentally induced changes in neuronal gene expression are a critical feature underlying behavioral plasticity. We have previously shown that transcription of the neuromodulator TGF-beta/DAF-7 is rapidly activated in the ASJ chemosensory neuron pair in response to the pathogenic bacteria Pseudomonas aeruginosa and that this expression change promotes avoidance behavior (Meisel, JD et. al., Cell, 2014). Using a genetically encoded calcium indicator we have also shown that specific secondary metabolites produced by P. aeruginosa can activate the ASJ neurons. To further understand the molecular mechanisms and genetic pathways responsible for transducing an environmental sensory input into a transcriptional response we performed a forward genetic screen to identify genes necessary for the activation of daf-7 transcription in response to P. aeruginosa. We have identified conserved genes involved in G protein-coupled receptor signaling and calcium signaling that are required for daf-7 activation in the ASJ neuron pair. We have also identified mutants with constitutive daf-7 expression in the ASJ neurons when C. elegans are propagated on non-pathogenic E. coli. Analysis of neuronal calcium dynamics in these mutant backgrounds will provide insight into how sensory activity and calcium signaling are coupled to rapid changes in transcription that modulate behavior.

Hilbert, Zoe et al. (2015) International Worm Meeting "Sexually-dimorphic Expression of a C. elegans Neuromodulator and Its Role in Behavior."

Hilbert, Zoe, Kim, Dennis

[International Worm Meeting, 2015]

Sex-specific differences in neuronal gene expression have been identified in a number of animal species, although the significance of these expression differences, in many cases, remains unclear. We have observed a sex-specific modulation in the expression pattern of the TGFß ligand, DAF-7, in male C. elegans. daf-7 is expressed normally in the ASI neuron pair and is known to regulate many aspects of C. elegans physiology, development and behavior. Recently, our lab has described the induction of daf-7 expression in an additional pair of chemosensory neurons, the ASJ neurons, in response to exposure to the pathogen Pseuodomonas aeruginosa and defined a role for this expression change in producing a pathogen avoidance behavior (Meisel, JD et. al., Cell, 2014). While hermaphrodites induce expression of daf-7 in ASJ in the presence of P. aeruginosa, we have observed that males exhibit daf-7 expression in the ASJ neuron pair in the absence of pathogen. This sexually dimorphic expression occurs stage-specifically in adult males, suggesting a possible role for this expression change in guiding male sexual behaviors or physiology. We have evidence to suggest that daf-7 expression may be involved in promoting male mate-searching/food leaving behavior and are currently working to further define the role of this sexually dimorphic expression pattern as it pertains to behavior. .

Hilbert, Zoe et al. (2015) International Worm Meeting "Natural Variation in Pathogen-induced Neuronal Gene Expression in C. elegans."

Kim, Dennis, Hilbert, Zoe, Meisel, Joshua

[International Worm Meeting, 2015]

We have been investigating natural variation in the neuronal expression of a TGF-ß signaling molecule, DAF-7, that plays a pivotal role in the regulation of diverse behaviors of C. elegans such as the dauer developmental decision, reproductive egg laying, and feeding. Normally expressed only in the ASI pair of chemosensory neurons, recent work in our lab (Meisel, JD et. al. Cell, 2014) has demonstrated that daf- 7 expression is induced in the ASJ neurons upon exposure to the bacterial pathogen, Pseudomonas aeruginosa, and that this change in expression is functionally important in producing a pathogen avoidance behavior. To investigate natural variation in this response to the microbial environment, we have examined a panel of 25 C. elegans wild isolate strains for differences in pathogen-induced daf-7 expression. We observe a wide range in the kinetics of daf-7 expression changes in the response of wild strains to pathogenic bacteria. Two strains were identified as having distinctly different phenotypes from N2: one which has constitutive expression of daf-7 in ASJ and another which only weakly induces daf-7 expression in response to P. aeruginosa .We have identified distinct genomic loci underlying these phenotypes and are currently working to define the natural polymorphisms that cause variation in the pathogen-induced neuronal expression of daf-7.

Kazumi Sakata et al. (2002) Japanese Worm Meeting "A Computer Simulation of the Nervous System of C. elegans"

Taro Ogurusu, Ryuzo Shingai, Tokumitsu Wakabayashi, Kazumi Sakata, Katsunori Hoshi

[Japanese Worm Meeting, 2002]

Computer simulation of the neural system of Caenorhabditis elegans was performed. The model neuron had Hodgkin-Huxley type voltage sensitive channels. Parameters of the channels were obtained by curve fitting to the electrophysiological experimental data (Pierce-Shimomura JT, et al. Nature. 410 , 694 (2001)). Chemical synapses were located within neurites and their transmission properties were set to be graded as those of Ascaris suum (Davis RE, Stretton AO. J Neurosci. 9 , 415 (1989)). Gap junction was reconstructed as the connection of neurite membranes between pre- and post-synaptic neurons. The following conditions were necessary to reconstruct realistic changes of membrane potentials; (1) the conductance of gap junction was lower than that of the mammal, (2) the time constants of the channels at chemical synapse was about 3 msec. When the synaptic delay, which included the whole time course from activation of presynaptic soma membrane to that of postsynaptic cell, was set to 500 msec, the whole network system showed periodic perturbation of membrane potential. However, this synaptic delay is very long and not realistic. Therefore, there should be some factors which make the practical delay long as 500 msec. Additionally, we discuss possibility of other factors to generate periodic activation of membrane potential, e.g., pacemaker-like neuron as observed in Ascaris (Angstadt JD, Stretton AOW. J Comp Physiol A. 166 , 165 (1989)).

Albritton, Sarah et al. (2011) International Worm Meeting "Regulation of X chromosome transcription in Caenorhabditis species."

Ercan, Sevinc, Albritton, Sarah

[International Worm Meeting, 2011]

Animals with different numbers of X chromosomes in males and females possess mechanisms to compensate for the difference in the X-linked gene dose between the two sexes. In addition to the X chromosome dose difference between the sexes, the presence of a single-copy X chromosome per two-copy diploid autosomes creates an important problem for males, because all X-linked genes are haploinsufficient compared to the autosomal genes. In C. elegans, hermaphrodites (XX) contain two Xes, whereas males (XO) contain a single X, therefore facing X haploinsufficiency. By performing microarray analysis of RNA abundance in XX and XO worms, we observed that the overall transcript levels from the X chromosome in both XX and XO animals is similar to that of overall expression from autosomes. This suggests that transcription from the single X in XO L3 hermaphrodites (TY2205, her-1(e1520) sdc-3(y126) V; xol-1(y9) X) is increased approximately two-fold. The mechanism of this upregulation is unclear. We had shown that the X chromosome promoters have higher GC content compared to the autosomes (Ercan et al 2010), suggesting a DNA-encoded mechanism of transcriptional regulation. We will study X upregulation by comparing transcription of orthologus genes that are on the X versus autosomes in four Caenorhabditis species. Ercan S, Lubling Y, Segal E, Lieb JD. High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans. Genome Res. 2011 Feb;21(2):237-44. PMID:21177966.

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

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

[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.

Andreia Teixeira-Castro et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Pathological ataxin-3 misfolding in C. elegans neurons"

Patricia Maciel, Richard Morimoto, Andreia Teixeira-Castro

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Expansion of polyglutamine (polyQ) tracts has been identified as the basis of at least nine neurodegenerative diseases, including Machado-Joseph disease (MJD). MJD is a hereditary ataxia of adult onset caused by expansion of a polyQ tract in ataxin-3 (AT3). AT3 is widely expressed and consists of an N-terminal globular domain with significant helical content, which spans the Josephin domain (JD), and a flexible C-terminal tail containing up to three Ubiquitin interacting motifs (UIM) and the polyQ tract. AT3-induced neurodegeneration affects a specific subset of neurons and is characterized by the presence of AT3- containing protein aggregates. Mutant AT3 forms mainly intranuclear inclusions in diseased human brain as well as in cell culture. Studies suggest that the pathological form of AT3 undergoes a conformational change leading to an alteration in protein homeostasis, misfolding and toxicity. To identify the factors involved in cell-specific pathogenesis observed for MJD, we generated pan-neural Caenorhabditis elegans models expressing chimeric fusion proteins of AT3, with normal and expanded polyQ lengths, tagged on the C-terminus with YFP. We are currently performing the behavioral analysis and looking at the aggregation properties of these models with particular emphasis on polyQ length-dependent aggregation and neurotoxicity. Once we have characterized our model, we will search for genetic modulators of AT3 pathogenesis thus revealing a subset of regulating genes uniquely relevant for mutant AT3 misfolding and toxicity in a metazoan. The comparison to the existing C. elegans polyQ models will contribute significantly in identifying the importance of protein context in cell-specific pathogenesis, providing a better understanding of the disease mechanisms.

Sobia Aslam et al. (2003) International Worm Meeting "T-box genes and the generation of morphological diversity"

Sara Maxwell, Jane Sowden, Alison Woollard, Lizzie Clarke, Sobia Aslam

[International Worm Meeting, 2003]

The T-box family of transcription factors is phylogenetically conserved and plays important roles during development. There are 20 T-box genes in C. elegans; more than in Drosophila, mice and humans. Most of the C. elegans T-box genes are highly diverged but one of these genes, mab-9(male abnormal), is an orthologue of human and murine Tbx20 and is also closely related to H15 in Drosophila. mab-9, was identified through mutation and functions to specify cell fate during hindgut and male tail development. In addition mab-9 plays a role in the developing nervous system. In mouse and humans, Tbx20 orthologues are expressed during early heart development and in the central nervous system. Vertebrate Tbx20 orthologues in zebrafish and chick show conserved expression patterns (1) and in Drosophila the Tbx20 orthologue, H15, is also expressed during early heart development (2) suggesting an important evolutionary role for Tbx20 in the specification of the heart. Hence, Tbx20 orthologues appear to have acquired diverse functions in chordates and nematodes, despite a common evolutionary origin. However it is also possible that there is an evolutionary parallel between heart and hindgut development. How far are the Tbx20 genetic pathways conserved between chordates and nematodes? We are investigating whether vertebrate Tbx20 can functionally substitute for mab-9 in vivo. Other potential phenotypic parallels in highly conserved T-box genes, for example tbx-2, are also being analysed. The other C. elegans T-box genes are highly diverged but nevertheless some of these have essential functions during development. tbx-23 appears to have no close orthologues within the metazoan phyla however, RNAi for tbx-23 resulted in highly penetrant embryonic lethality. Examination of animals escaping embryonic arrest revealed severe posterior defects, suggesting that tbx-23 may play a role in posterior patterning during embryogenesis. Reporter gene analysis for tbx-23 expression is in progress. 1) Meins et al., Genomics 76, 317-332 (2000) 2) Griffin et al., Dev. Biol 218, 235-247 (2000)