Kyuhyung Kim et al. (2005) International Worm Meeting "Specification of chemosensory neuron identity by the C. elegans UNC-3 Olf/EBF protein"

Kyuhyung Kim, Helen Yeung, Marc Colosimo, Piali Sengupta

[International Worm Meeting, 2005]

Neuronal identities are specified by the combinatorial functions of transcription factors. In many cases, transcription factor codes act to not only promote cell type-specific characteristics, but to also repress inappropriate gene expression. Although individual components of these codes have been identified in several systems, the mechanisms by which they act are not yet fully understood. Members of the well-conserved Olf/EBF (O/E) transcription factor family have been shown to play important roles in neuronal and non-neuronal development and differentiation. O/E proteins are highly expressed in the olfactory epithelium, and O/E binding sites have been identified upstream of olfactory genes. However, the roles of O/E proteins in sensory neuron development are unclear. The C. elegans genome is predicted to encode a single member of the O/E family, UNC-3. Intriguingly, unc-3 is expressed in a single chemosensory neuron type the ASI neurons, although the role of unc-3 in ASI neuron development is unclear. We found that expression of ASI-specific genes including the daf-7 TGF- gene is downregulated in unc-3 mutants, and the ASI neurons misexpress terminal differentiation genes specific to multiple neuron types. In addition, misexpression of UNC-3 in other sensory neurons is sufficient to repress neuron-specific gene expression. We identified putative UNC-3 binding sites in the regulatory regions of affected genes using via bioinformatics and promoter deletion experiments and showed that UNC-3 directly binds these sequences to mediate repression. Thus, UNC-3 is a crucial member of the transcription factor repertoire that specifies an ASI identity. These findings suggest that O/E proteins may function via similar activation and repression mechanisms to regulate cell-specific features in the olfactory and other tissues in vertebrates. We searched for additional transcription factors required for specification of ASI identity. Based on promoter analysis and bioinformatic searches, we putatively identified a cis-regulatory element that activates gene expression in the ASI neurons. Currently we are further testing whether this ASI motif is neccesary and/or sufficient to promote all ASI neuron-specific gene expression and plan to identify the trans-acting factors binding this sequence.

Mayumi Shibuya et al. (2005) International Worm Meeting "Plasticity in chemosensory neuron-specific gene expression and behavior"

Helen Yeung, Piali Sengupta, Mayumi Shibuya, Kyuhyung Kim

[International Worm Meeting, 2005]

Plasticity in sensory responses enables animals to appropriately modulate their behaviors and developmental programs in response to constantly changing environmental conditions. In addition to acute neuronal plasticity, cues experienced during specific developmental stages are critical in shaping adult behaviors. The nematode C. elegans responds to conditions of overcrowding, limited food and high temperature by arresting development as a dauer larvae. A primary signal triggering entry into the dauer stage is high levels of secreted dauer pheromone. We and others showed previously that exposure to dauer pheromone at any developmental stage reversibly represses the expression of olfactory receptor genes in the ASI chemosensory neurons, suggesting that acute modulation of chemoreceptor expression by environmental cues may be a mechanism by which C. elegans can rapidly alter its sensory behaviors. We have now shown that similar to the chemoreceptor genes, expression of additional signaling molecules in chemosensory neurons is also similarly affected by exposure to dauer pheromone. Interestingly, we have also found that exposure to low levels of dauer pheromone only at early developmental stages appears to be sufficient to irreversibly alter a subset of gene expression in the chemosensory neurons. Moreover, adult animals exposed to dauer pheromone at early stages or animals that have transiently passed through the dauer stage exhibit sensory behaviors that are distinct from those of animals that have not experienced these environmental and developmental conditions. These results suggest that the gene expression pattern in sensory neurons and sensory behaviors may reflect a memory of the animals developmental and environmental history. To identify the signaling pathways and genes required for environmental and developmental plasticity, we performed a genetic screen utilizing dauer pheromone-mediated downregulation of chemoreceptor gene expression in the ASI neurons. In a genetic screen of ~40,000 haploid genomes, we isolated several mutants which are currently being further mapped and characterized.

Kyuhyung Kim et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Daumone regulates expression of a subset of chemoreceptor genes via a CaMK cascade"

Helen Yeung, Piali Sengupta, Mayumi Shibuya, Kyuhyung Kim

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

Animals must be able to alter their development and behavior in response to changing environmental conditions and on the basis of prior experience. This developmental and behavioral plasticity is mediated by changes in gene expression. Our goal is to understand the mechanisms by which environmental signals are transduced and integrated to modulate changes in sensory gene expression, and ultimately, in behavior. C. elegans responds to conditions of overcrowding, limited food and high temperature by arresting development as a dauer larva. A primary signal triggering entry into the dauer stage is high levels of secreted dauer pheromone. We and others showed previously that exposure to low levels of crude dauer pheromone alters the expression of a subset of putative G-protein coupled chemosensory receptor genes in the ASI chemosensory neurons (Nolan et al., 2002; Peckol et al., 1999). We have now shown that similar to crude dauer pheromone, low-levels of chemically synthesized dauer pheromone (daumone: Jeong et al., 2005) reversibly repress expression of the str-3 chemoreceptor gene in ASI. Repression of str-3 expression upon low levels of daumone exposure can be initiated only at the L1 stage, but expression is restored upon removal of daumone at any developmental stage. To identify the signaling pathways and genes required for this environmentally-regulated gene expression, we performed a genetic screen as well as a candidate gene search. We identified cmk-1 (Ca2+/calmodulin-dependent protein kinase I :CaM Kinase I) and ckk-1 ( CaM kinase kinase) as genes that are required to downregulate str-3 expression upon daumone exposure. In addition, we tested crh-1 (cyclic AMP response element binding protein :CREB) ,a putative target of the CaMK cascade and showed that crh-1 is required for str-3 expression even in the absence of daumone. Thus, the CAMK cascade may act to inhibit CRH-1 function upon daumone exposure. Surprisingly, neither cmk-1 nor crh-1 appear to act cell-autonomously to regulate str-3 expression in the ASI neurons. Currently, we are investigating the spatial requirements of cmk-1 and crh-1 function in the regulation of str-3 expression. We are also cloning additional genes identified in our genetic screen, and identifying the cis-regulatory elements required to confer daumone-mediated regulation.

James P McCarter et al. (2001) International C. elegans Meeting "Genes from Other"

Michael Dante, James P McCarter, John C Martin, Deana Pape, Brandi Chiapelli, Todd N Wylie, Robert H Waterston, Sandra W Clifton

[International C. elegans Meeting, 2001]

To build upon knowledge gained from the genome of C. elegans , we have begun generating Expressed Sequence Tags (ESTs) from parasitic (and free-living) nematodes. This project will generate >225,000 5' ESTs from 14 species by 2003. Additionally, the Sanger Centre and Edinburgh Univ. will complete 80,000 ESTs from 7 species. Through these combined efforts, we anticipate the identification of >80,000 new nematode genes. At the GSC, approximately 35,000 ESTs have been generated to date including sequences from Ancylostoma caninum, Heterodera glycines, Meloidogyne incognita and javanica, Parastrongyloides trichosuri, Pristionchus pacificus, Strongyloides stercoralis and ratti, Trichinella spiralis, and Zeldia punctata . We will report on our progress in sequence analysis, including the creation of the NemaGene gene index for each species by EST clustering and consensus sequence generation, identification of common and rare transcripts, and identification of genes with orthologues in C. elegans and other nematodes. All sequences are publicly available at www.ncbi.nlm.nih.gov/dbEST. NemaGene sequences and project details are available at WWW.NEMATODE.NET. We would like to thank collaborators who have provided materials and ideas for this project including Prema Arasu, David Bird, Rick Davis, Warwick Grant, John Hawdon, Doug Jasmer, Andrew Kloek, Thomas Nutman, Charlie Opperman, Alan Scott, Ralf Sommer, and Mark Viney. This work is funded by NIH-AI-46593, NSF-0077503, and a Merck / Helen Hay Whitney Foundation fellowship.

W.K. So et al. (2006) European Worm Meeting "Sex pheromone production in dioecious nematodes and the perception pathway delineated by genetics"

W.S. Yeung, M.Y. Wu, C.M. Chan, W.K. So, K.L. Chow

[European Worm Meeting, 2006]

W.K. So, C.M. Chan, M.Y. Wu, W.S. Yeung and K.L. Chow. In dioecious nematodes, finding the mating partner and successful mating is instrumental to their reproductive success. To ensure these tasks are accomplished, sex pheromone production is a common strategy adopted in the nematode world. In Caenorhabditis remanei, a dioecious species, a sex-specific attractant was identified to be produced by females for attracting males. In this study, we focus on four areas of characterization, the properties of the pheromone, its regulatory production in female, male perception and species specification. In the characterization part, our GC-MS analysis result identifies two candidate chemicals in the samples, the exact identity of which will be confirmed by chemical synthesis coupled with bioassays. These attractant substances are produced under tight developmental control in the gonad as confirmed by laser ablation of gonadal progenitor cells in different combination. Their activity could be abolished by mating with males. Based on our pervious results, the males of the androdioecious can be attracted by this sex pheromone. So for the male perception part, we employed males from various Caenorhabditis elegans mutants with defects in cell lineage or molecular function to delineate the perception pathway. Our results show that two different sensory neurons and an interneuron are required. Moreover, a signaling process dependent of G protein coupled receptor is crucial for the pheromone perception, where modulation by other molecular components has been observed. Hypothesis suggesting a conservation of the pheromone perception pathway across different species will be tested using chemo-attraction assay on con-specific and hetero-specific combination of testing animals. (The project is funded by Research Grant Council, Hong Kong.)

Todd N Wylie et al. (2001) International C. elegans Meeting "NEMATODE.NET, a Tool for Navigating Sequences from Parasitic and Free-living Nematodes"

Todd N Wylie, John C Martin, Robert H Waterston, James P McCarter, Sandra W Clifton

[International C. elegans Meeting, 2001]

Two web sites have been established to allow easier access to nematode sequences from species other than C. elegans and C. briggsae ; WWW.NEMATODE.NET is maintained by the Genome Sequencing Center (GSC) at Washington University in collaboration with North Carolina State University, and WWW.NEMATODES.ORG is maintained by Mark Blaxter's lab at the University of Edinburgh. Useful features being built for NEMATODE.NET include the following - 1) Searches: All nematode expressed sequence tags (ESTs) generated at the GSC, currently 32,000 from 10 species, and NemaGene clusters built from these ESTs, are available for BLAST and text searching. Searches can be directed by species, library, or nematode clade in a way that is not possible using the NCBI EST database dbEST. 2) FTP: All EST project data can be downloaded for local analysis including FASTA files and sequence trace image files. 3) Trace Viewer: Fluorescent trace representations for each EST can be viewed. Traces can sometimes provide additional sequence information not included in the EST due to quality value cut-offs. 4) Project Updates: Information is available about libraries in construction and sequencing in progress as the project expands toward 235,000 ESTs. 5) Clone Requests: Details on clone availability and ordering procedure are provided. 6) Links: The site includes an up-to-date set of 300 links to information on human, animal, and plant parasitic nematodes. Further plans for NEMATODE.NET include linking of ESTs to their closest C. elegans homologues by DAS third-party curation of Wormbase. This work is funded by NIH-AI-46593, NSF-0077503, and a Merck / Helen Hay Whitney Foundation fellowship.

Schein JE et al. (1996) West Coast Worm Meeting "CONSTRUCTION AND UTILIZATION OF THE CHROMOSOME IV COSMID TRANSGENIC LIBRARY"

Schein JE, Rose AM, Baillie DL, Franz NW, Janke DL

[West Coast Worm Meeting, 1996]

Construction of a chromosome IV cosmid transgenic library of C. elegans strains carrying sequenced cosmids in extrachromosomal arrays has begun as part of our labs' collaboration with the Genome Sequencing Consortium (see abstract by D. Janke et al.). There are 20 chromosome IV cosmids available in transgenic strains at the time of this writing, and we estimate a total of 40-50 will be available to the community by the end of July. In order to enhance the utility of this library, we are also using these strains to localize approximately 50 essential gene mutations, previously identified in the Baillie lab, to cosmids by way of transgenic rescue experiments (for results of similar efforts on chromosomes III and V, see abstracts by Nigel O'Neil et al., Helen Stewart et al., and Brad Barbazuk et al.). Correlation of the genetic and physical maps in this manner will facilitate the selection of candidate cosmids for use in further rescue experiments, and thereby increase the speed with which the connection between genetic mutations and DNA sequence is made. Currently these rescue experiments are focused in the sDf2 region, in particular in the lin-3 - let-60 interval, which contains 13 essential gene mutations and 21 overlapping cosmids. The two genes bracketing this interval, lin-3 and let-60(ras), were previously placed on the physical map by others. To date, two additional essential genes have been positioned to cosmids on the physical map; let-70(ubc2) (in collaboration with Mei Zhen and Peter Candido) and let-64. It is anticipated that by the time of the meeting several additional mutations will have been positioned to cosmids. This work is being funded by a grant from the Canadian Genome and Associated Technologies Program to Ann Rose and David Baillie.

Hartman PS et al. (1991) International C. elegans Meeting "SOME RAD HAPPENINGS FROM FORT WORTH."

DeWilde D, Reddy J, Hartman PS

[International C. elegans Meeting, 1991]

1. UV radiation mutagenesis in C. elegans: Helen Stewart and David Baillie have noted that many UV radiation-induced mutations are chromosomal rearrangements rather than point mutations. We have begun to extend this observation by isolating UV radiation-induced mutations in fem-3 and unc-22. As suggested to us by Judith Kimble, and as will be described at the meeting,fem-3 is particularly well suited for these studies. Mutations have been selected at low and high radiation fluences, as well as in both rad(+) and rad(-) genetic backgrounds. These are currently the subject of both genetic and molecular analyses, with the ultimate hope of elucidating specific sequence contexts which are misrepaired into deficiencies. 2. Cloning a rad gene: We have isolated two allelic mutations in the high-hopper strain TR679 which confer hypersensitivity to methyl methanesulfonate and UV radiation. They map in between unc-36 and dpy-17 on linkage group III. Each mutation was crossed 12 times into an N2 background, several of which including forced recombinations in between the mutations and either unc-36 or dpy-17. Southern blots were performed, using Tcl as a probe (kindly provided by Phil Anderson) and revealed one novel Tc l- containing fragment in one mutant and three in the other. We are currently cloning these. Since both mutants are hypersensitive only during embryogenesis, we look forward to performing Northern blot analyses in order to determine if this developmental regulation of DNA is exercised at the transcriptional level. 3. The rad mutants are not hypersensitive to 8-methoxypsoralen (8MOP) phototreatment. Extending the observations of Fujita and coworkers (Photochem. Photobiol. 39, 831), survival of three staged populations N2 and four rad mutants was determined after 8-MOP phototreatment. As opposed to their responses after exposure to other DNA-damaging agents, none of the four were hypersensitive. Split-dose experiments indicated that DNA-DNA crosslinks were primarily responsible for lethality. Little if any crosslink repair was observed in N2 using two different assays, explaining why the rad mutants were not hypersensitive to 8-MOP inactivation.

Stewart HI et al. (1996) West Coast Worm Meeting "Recessive Lethal Mutations and Deficiency Analysis in Caenorhabditis elegans on LGIII(left) and Balanced by sDp3"

Vatcher GP, Ha TT, Janke DL, Gilchrist EJ, Stewart HI, Baillie DL

[West Coast Worm Meeting, 1996]

We have isolated 202 new, EMS induced, recessive lethal mutations on the left arm of LGIII. The free duplication sDp3 (III;f) was used to rescue recessive mutations linked to dpy-17 (recovery rate 1.9%). Of these 202 mutations, 109 have been characterized and analysis of the remaining mutations is in progress. A subset, of 62 recessive lethals, are also marked with ncl-1 and will facilitate the mosaic analysis of these lethal mutations. We have identified 82 new essential genes let-700 to let-790. Of these new essential genes, 13 have multiple alleles, thus we estimated that we have only a 28% saturation of the area for essential genes. As there is about 4.8 Mb of DNA, in the interval between unc-93 and dpy-19, we estimate that there will be approximately 293 essential genes, in this region. We intend to generate another set of recessive lethal mutations to facilitate saturation of this area for essential genes. Both gamma rays and ultraviolet light were used to generate a set of overlapping deficiencies. We have shown that duplications can be used to rescue large deletions such as sDf121, sDf125, sDf127 and sDf130, as well as smaller deficiencies. This indicates that there are no "holes" or lethal mutations along the length of sDp3, in the areas uncovered by these large deficiencies. We have shown that duplications, such as sDp3, are valid and useful as balancer systems and have utilized deficiency mapping to resolve the genetic mapping of essential genes. Selected visible mutations have also been complementation tested with our deficiencies. PCR analysis was utilized, with these deficiencies, to correlate the genetic and physical maps and to further define the end points of these deficiencies ( See poster ; Norm Franz et al, this session). As well, work has been progressing on the rescue of essential genes (See poster; Helen Stewart et al, this session), utilizing transgenic strains constructed in this laboratory (see presentation by Diana Janke et al, this session). This work has been funded by a grant from NIH (National Institute of Health), U.S.A. to Ann. Rose; D. Riddle and D.L. Baillie.

Helen Sharpe et al. (2006) European Worm Meeting "3' End Processing and Transcription Termination in the Nematode Caenorhabditis elegans"

Helen Sharpe, Andre Furger, Cathy Browne

[European Worm Meeting, 2006]

Helen Sharpe, Cathy Browne and Andr Furger. . We are using C. elegans to study the relationship between the recognition of the poly(A) sites and transcription termination in a chromosomal context. The compact genome, trans-splicing and the arrangements of genes in operon-like structures make C. elegans an interesting system to study RNA polymerase II transcription termination. In both mammals and yeast the recognition of a functional poly(A) site is instrumental in directing transcription termination. However, when operons in C. elegans are transcribed as polycistronic pre-mRNAs, several poly(A) sites are transcribed and recognized without inducing transcription termination. This implies that the two processes in the nematode can either be uncoupled, or that specific mechanisms are in place to prevent termination despite the recognition of the poly(A) sites. In addition, trans-splicing may require a more stringent transcription termination control in order to prevent expression of downstream located genes that are accidentally transcribed.. To address this issue, we began to analyse the relationship between the recognition of poly(A) sites and transcription termination in three nematode genes. We have used a RT-PCR based technique to analyse how far transcription continues into the 3 flanking region of three C. elegans genes before cleavage at the processing site occurs.. We first investigated the transcription profile at the 3 end of vit-2, a gene that undergoes conventional splicing (removal of intronic sequences) but is not trans-spliced. We detected uncleaved contiguous pre-mRNA transcripts that contained sequences located at least 900 bases downstream of the poly(A) site. As has been shown in a mammalian system, these extended pre-mRNAs still contain the terminal intron sequences. Transcription of the vit-2 gene results in a contiguous pre-mRNA that extends into a downstream positioned predicted ORF located on the anti-sense strand and extends into promoter sequences of a downstream positioned predicted gene on the sense strand.. We are currently comparing the vit-2 transcription profile to transcriptional events in several trans-spliced genes. Additionally, we are developing nuclear run-on analysis to confirm our transcription profiles.