Chris R Gissendanner et al. (2003) International Worm Meeting "Expression and function of the NGFI-B nuclear receptor in C. elegans and Dirofilaria immitis."

Ann Sluder, Chris R Gissendanner, George Tzertzinis, Cathy Shea, Claude Maina, Jianping Xiao

[International Worm Meeting, 2003]

We are interested in the biological roles of nuclear receptor (NR) transcription factors in filarial nematodes, specifically those that may regulate larval transitions during the filarial parasitic life cycle. Our lab has cloned several NR genes from the filarid nematode Dirofilaria immitis, also known as dog heartworm, that are homologs of NR genes that regulate molting and metamorphosis in insects. One such NR gene is dinhr-2, the D. immitis homolog of DHR38 from Drosophila and NGFI-B from vertebrates. C. elegans also encodes a DHR38/NGFI-B homolog, nhr-6. We are performing a comparative molecular and biochemical analysis of nhr-6 and dinhr-2 as well as assessing biological functions of nhr-6 in C. elegans. Phenotypic analysis of a nhr-6 mutant reveals that nhr-6 is required for the normal ovulation of oocytes. nhr-6 is expressed in the developing spermatheca and nhr-6(lg6001) mutants have malformed spermathecas, as demonstrated by cell marker analysis. Spermathecal function is disrupted such that oocytes are fragmented during ovulation, leading to formation of abnormal eggs and embryonic lethality. The spermathecal development role of nhr-6 indicates that NGFI-B function in C.elegans has diverged from the molting function in insects. However, many of the biochemical functions of NGFI-B are conserved in NHR-6 as well as DiNHR-2. These include phosphorylation at a conserved Akt site and heterodimerzation with a RXR nuclear receptor, a common NR heterodimer partner in insects and vertebrates. The RXR heterodimerization property of NHR-6 is conserved despite the absence of a RXR homolog in C. elegans. We are currently assessing the biological relevance of NHR-6 phosphorylation and heterodimerization in C. elegans using rescue assays. In addition, we are exploring the temporal and spatial expression of dinhr-2 during the D. immitis life cycle and during molting to assess whether NGFI-B function in filarids is more similar to NGFI-B function in Drosophila or C. elegans. Results of these experiments will be presented.

John Peloquin et al. (2007) International Worm Meeting "Worms and Filthy Lucre: Applying C. elegans to natural product development in industry."

Stuart Reeves, John Peloquin

[International Worm Meeting, 2007]

Diamond V Mills is a medium sized Agricultural Biotechnology company with more than 63 years of success in producing dietary supplements and ingredients. Recently the company has moved into human nutrition through its formation of Embria Health Sciences. Thus, the product repertoire has increased and is increasing. Our products arise naturally as the result of subtle alterations of fermentative processes, which are still not completely understood. Furthermore, their effects on animal biology are pleotropic. This presents some serious economic challenges in that far more product prototypes can be produced than economically tested in vertebrate models to demonstrate efficacy. Because many biochemical and signal transduction pathways are conserved between C. elegans and other animals, we have begun to develop C. elegans-based medium through-put screens to identify new product prototypes. We present preliminary data on anti-aging effects of an Embria product as an example of our future goals. (this work was initiated with technical assistance from Mark A. Wilson and Cathy Wolkow of the NIA).

Sluder AE et al. (1995) International C. elegans Meeting "NEMATODE NUCLEAR HORMONE RECEPTORS: TWO DOZEN AND COUNTING"

Maina CV, Lindblom TH, Hough DM, Sluder AE, Wong S, Robbins A

[International C. elegans Meeting, 1995]

To date 24 C. elegans genes encoding members of the nuclear hormone receptor (NHR) superfamily of transcription factors have been identified, primarily by our direct homology screens and by the genome sequencing project. Most of these genes encode widely divergent members of the NHR superfamily. While all exhibit significant sequence similarity in the highly conserved DNA-binding domain, many (13/24) have unique amino acid sequences in the region responsible for DNA binding site recognition, suggesting that the DNA binding specificities of these C. elegans proteins will differ from those of previously characterized NHRs. All of the C. elegans NHRs are "orphan" receptors for which the ligands, if any, are unknown. Though not as highly conserved as the DNA-binding domains, blocks of sequence similarity are found in the ligand-binding domains of all NHRs known to bind ligands. These regions of similarity are also seen in many, but not all, orphan receptors, including at least 10 of the C. elegans NHRs. Five NHR genes have also been identified in the dog heartworm Dirofilaria immitis. One of these, dinhr-2, is closely related to the C. elegans gene nhr-6. Two others exhibit significant homology to the two genes encoding subunits of the Drosophila ecdysone receptor (see abstract by Shea et al.). As little is known about the biological roles of most of the C. elegans NHR genes we are undertaking genetic analysis of a selected subset. Tc1 insertions have been generated in nhr-6 and in the embryonically expressed nhr-2; deletion screens are underway. Progress in these and other screens will be presented.

Julianne Paille et al. (2007) International Worm Meeting "An RXR nuclear receptor homolog from Pristionchus pacificus."

Sharita Flowers, Chris R. Gissendanner, Julianne Paille

[International Worm Meeting, 2007]

The retinoic acid X receptors (RXRs) are considered to be an ancient group of nuclear receptors and have been identified in a variety of metazoans, including nematodes. However, the presence of RXR homologs in nematodes has only been confirmed in filarial parasites (Shea et al, 2004). In insects, the RXR homolog USP interacts with the EcR nuclear receptor to function as the holoreceptor for the molting hormone 20-hydroxyecdysone. Neither RXR nor EcR nuclear receptor homologs are encoded in the C. elegans and C. briggsae genomes, even though many of the nuclear receptors that function downstream of USP/EcR in insects are found in C. elegans and function to regulate the nematode molting process (Gissendanner et al, 2004). Therefore, an interesting question is whether RXR, and EcR, are found in other, non-parasitic nematodes. A search of genome sequences from the free-living nematode Pristionchus pacificus identified a genomic fragment encoding a potential RXR nuclear receptor. This fragment was used to design RT-PCR experiments to isolate cDNAs encoding this putative RXR nuclear receptor. We have successfully isolated several full-length cDNAs corresponding to the genomic sequence, and these cDNAs encode a nuclear receptor with significant similarity to RXR nuclear receptors (>80% in the DNA binding domain). Several mRNA isoforms have been isolated. Temporal expression profiles using semi-quantitative RT-PCR indicate expression fluctuations that correlate with the molting cycle, suggesting a role for the Pristionchus pacificus RXR during molting. We are currently further testing this hypothesis using Northern analysis and RNA interference. The isolation of an RXR nuclear receptor from a free-living nematode provides an opportunity to investigate the biological functions and evolution of RXR in nematodes.

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.

Masahiro Tomioka et al. (2002) Japanese Worm Meeting "The insulin-like signaling pathway participates in learning in salt chemotaxis"

Hirofumi Kunitomo, Yuichi Iino, Masahiro Tomioka

[Japanese Worm Meeting, 2002]

We have previously reported a form of learning in which worms starved for several hours in the presence of salt such as NaCl (food-/salt+ conditions) learn to avoid the salt, which is otherwise chemoattractive to worms. This dramatic change in the chemotaxis behavior is not induced under the food+/salt+ or food-/salt- conditions (Saeki, Yamamoto and Iino, 2001). Several mutants of daf-2, which encodes an insulin/IGF-I receptor-like protein, and the hx546 mutant of age-1, which encodes a PI-3-kinase acting downstream of DAF-2, shows defects in this form of learning at 25C: they still shows positive chemotaxis after food-/salt+ conditioning. We found that INS-1, one of a multitude of insulin-like peptides predicted in C. elegans, is also involved in the plasticity of chemotaxis. The deletion mutant of ins-1, ins-1(nr2091), shows a defect in this paradigm. This observation is intriguing given the results of Pierce et al. (2001) that this mutant shows no dauer phenotype nor affects life span, while overexpression of ins-1 causes a weak Daf-c phenotype by itself and enhances the Daf-c phenotype of daf-2 when combined with the daf-2 mutations. A genomic clone for the ins-1 gene rescued the learning defect when introduced into the ins-1(nr2091) mutant at a low dose. When introduced at a higher dose, however, the defect was not rescued. We are characterizing the overexpression phenotype in more detail. The mg305ts mutant of age-1 also shows a defect in learning at 25C. This learning defect of age-1(mg305) was rescued by neuronal expression of age-1(+) from the pan-neuronal unc-14 promoter. In contrast, expression of age-1(+) in the body-wall muscle from the unc-54 promoter or in the intestine from the ges-1 promoter was not sufficient to rescue the defect. These results suggest that the insulin-like ligands act on neurons and affects plasticity of chemotaxis as well as dauer formation and longevity, possibly in a different way. We are in the process of further narrowing down the site of age-1 action for the chemotaxis learning. We thank Cathy Wolkow for the cell-type restricted age-1 strains, and Gary Ruvkun for the ins-1(nr2091) strain.

Ewbank JJ et al. (1995) International C. elegans Meeting "TIME FOR SOME RESULTS: MOLECULAR CHARACTERISATION OF THE CLK-1 LOCUS."

Hekimi S, Ewbank JJ, Barnes TM, Lakowski B

[International C. elegans Meeting, 1995]

Mutation in the clk-1 locus disrupts the normal temporal control of a wide range of developmental and behavioral events; see Wong et al., Genetics 139:1247-1259 (1995). Perhaps fittingly for clk-1, progress towards the gene's cloning has been slow (wm93p124; ecwm94p77). Injection of cosmid pools spanning the candidate region has failed to produce rescue. RFLP mapping has been hampered by an apparent paucity of polymorphisms between N2, RC301 and RW7000 strains in the region. The strategy of refining the likely position of clk-1 by locating the left- and right-flanking genes dpy-17 and lon-1 has also proved problematic. Extensive Southern analyses have failed to reveal any allele-specific polymorphisms (thanks to Cathy Savage for lon-1 strains). Further, for lon-1, the usual co-injectable marker pRF4 on its own gives phenotypic rescue (wild-type length rollers; presumed to be a indirect effect), while arrays covering the supposed location of dpy-17 produce a dominant Dpy effect, potentially confounding detection of any rescue. A 32P non-complementation screen has generated new alleles of clk-1, dpy-17 and lon-1. These will be used in another round of Southern analyses in the hope of uncovering allele-specific polymorphisms. One positive lead that we are pursuing involves gro-1(e2400); see wbg11#5p60; ecmw92p5. The gro-1 locus lies close to clk-1; by scoring Sma non Dpy in the six factor cross dpy-17(e164)gro- 1(e2400)sma-4(e729)/ unc-79(e1030)clk-1(e2519)lon-1(e185) it has been positioned 0.03 cM (approx. 15 kb) to the left of clk-1. Two spontaneous mutants (bli-1(qm49) II and dpy-7(qm63) V) have been recovered from gro-1 strains, implicating the activity of a transposon which may be directly associated with the gro-1 mutation. As dpy-7 has been cloned, this may give us an indirect route to speed the cloning of both gro-1 and clk-1. The progress towards identifying clk-1, gro-1, dpy-17 and lon-1 will be reported.

Marisa L Foehr et al. (2003) International Worm Meeting "The C. elegans Schnurri homolog SMA-9 mediates cell type specific LIN-12/Notch signaling in the postembryonic mesoderm."

Yuan Jiang, Marisa L Foehr, Maegan Rivard, Rachel Fairbank, Jun Liu

[International Worm Meeting, 2003]

Many developmental processes including the specification of cell fate are mediated by highly conserved cell-cell signaling pathways. While the general mechanisms by which these signaling pathways function are well defined, the mechanisms mediating how the same signaling pathway can regulate different processes in different cellular context are less well understood. We are interested in a molecule, SMA-9, that functions specifically with the Notch signaling pathway to regulate cell fate specification in the C. elegans postembryonic mesodermal lineage, the M lineage. In the M lineage, sma-9 mutants specifically affect the decision between two cell fates, the sex myoblasts and the coelomocytes. In addition, sma-9 mutant animals are smaller than wild type. Work from the lab of Cathy Savage-Dunn has shown that sma-9 encodes the C. elegans homolog of the Drosophila protein Schnurri, a known regulator of the TGFb signaling pathway. The Savage-Dunn lab has also shown that SMA-9 does interact with the TGFb signaling pathway in regulating body size. However, the function of SMA-9 in M lineage fate specification appears to be independent of the TGFb signaling pathway. We have shown that sma-9 genetically interacted with LIN-12/Notch specifically in the M lineage. In the yeast two-hybrid system, SMA-9 interacted with LAG-1, a downstream transcription factor of the Notch signaling pathway. These suggest that SMA-9 might mediate different signaling pathways in a cell-type specific manner. Consistent with this, SMA-9 has different splicing isoforms. Therefore, we hypothesize that the different SMA-9 isoforms might mediate different signaling pathways in different cell types. To test the above hypothesis, we have generated isoform specific antibodies to two of the three SMA-9 isoforms and examined the expression pattern of SMA-9 using these antibodies. Both SMA-9 isoforms are present in the nucleus. However, they showed different patterns of expression. We are further characterizing the function of each isoform in the M lineage by examining their expression in the M lineage and by testing their interaction with LAG-1 in solution and in worm extracts. A suppressor screen for sma-9 has also been carried out in an attempt to isolate other factors that interact with SMA-9.

Masahiro Tomioka et al. (2003) International Worm Meeting "The insulin-like signaling pathway participates in learning in salt chemotaxis"

Yuichi Iino, Masahiro Tomioka, Hirofumi Kunitomo

[International Worm Meeting, 2003]

We have previously reported a form of learning in which worms starved for several hours on a plate containing salt such as NaCl (food-/salt+ conditions) learn to avoid the salt, which is otherwise chemoattractive to worms. This dramatic change in the chemotaxis behavior is not induced under the food-/salt- or food+/salt+ conditions (Saeki et al. 2001). A similar change of chemotaxis is induced by conditioning in a salt-containing buffer.We found that the insulin-like signaling pathway, which is known to regulate dauer formation, longevity and fat metabolism, participates in this form of learning. Several mutants of daf-2, which encodes an insulin/IGF-I receptor-like protein and mutants of age-1, which encodes a homologue of phosphoinositide 3-kinase, showed defects in learning. The loss-of-function mutant of pdk-1, which encodes a homologue of 3-phosphoinositide-dependent-kinase, also showed small but significant defect in learning. These data suggest that AGE-1 and possibly PDK-1 act downstream of DAF-2.Of a multitude of insulin-like peptides predicted in C.elegans, a deletion mutant of ins-1, ins-1(nr2091), showed a defect in this paradigm. On the other hand, a Daf-c mutant of daf-28, encoding a proposed insulin-like ligand for dauer formation, did not show an abnormality. Expression of ins-1(+) at the adult stage using the heat-shock promoter rescued the learning defect of the ins-1 mutant. Therefore the action of ins-1 at the adult stage is required for this form of learning.To determine where this signaling works, we expressed age-1 cDNA using cell-specific promoters and tested which transgenes rescued the learning defect of the age-1 mutant. Expression in ASE neurons, which are the main salt-sensing chemosensory neurons, using the gcy-5/7 promoters or in all chemosensory neurons except for ASE, using the odr-4 promoter, rescued the learning defect of the age-1 mutant. However, expression in several interneurons (AIZ and AIB etc.) using the odr-2 pomoter did not rescue the learning defect. These data suggest that the insulin-like ligand acts on the receptors in various chemosensory neurons including ASE.We thank Cathy Wolkow for the age-1 cDNA, Gary Ruvkun for the ins-1(nr2091) strain, Takeshi Ishihara for the H20 promoter and Yasumi Ohshima for the unc-14 promoter.

Masahiro Tomioka et al. (2005) International Worm Meeting "Regulation of salt chemotaxis learning by the insulin-like signaling pathway in C. elegans"

Hirofumi Kunitomo, Yuichi Iino, Masahiro Tomioka

[International Worm Meeting, 2005]

The chemotaxis of C. elegans is altered by past experience. The animals are attracted to NaCl, but show avoidance behaviors after a conditioning with NaCl and starvation (food-/NaCl+). This behavioral plasticity is not induced under the food-/NaCl- or food+/NaCl+ conditions, and therefore is likely to be a form of associative learning (1). The insulin-like signaling pathway participates in this form of learning, which we call salt chemotaxis learning. Several mutants in the insulin-like signaling pathway such as mutants of ins-1, daf-2 and age-1, which encode homologues of insulin peptide, insulin/IGF-I receptor and PI 3 kinase, respectively, showed learning defect: they show attractive behaviors to NaCl even after the food-/NaCl+ conditioning (2). We investigated the site of action of AGE-1 by cell-specific rescue experiments. Expression of age-1(+) in ASE, main salt-sensing neurons, rescued the learning defect of the age-1 mutants. Interestingly, the learning defect was rescued by the expression only in ASER, the right member of ASE neurons, although the expression in ASEL, the left member of ASE neurons, ASI, ASG and ADF, the other salt-sensing neurons or a subset of interneurons including AIY and AIZ did not rescue the defect. Therefore AGE-1 mainly acts in the ASER neuron in the salt chemotaxis learning. Chemotaxis to Cl-, which is mainly received by ASER neurons is most affected in salt chemotaxis learning, while chemotaxis to Na+, which is mainly received by ASEL neurons is only weakly altered, consistent with AGE-1 mainly acting in the ASER neuron. A functional INS-1 reporter construct, in which INS-1 is translationally fused to green fluorescent protein, is expressed mainly in AIA, a pair of interneurons that receive inputs from many sensory neurons. The learning defect of the ins-1 mutant was rescued by the expression in several neurons including AIA using the gpa-2 promoter, although the expression in chemosensory neurons using the odr-4 promoter or a subset of interneurons including AIY and AIZ using the ser-2 promoter did not rescue the defect. Animals in which AIA neurons were killed by laser microsurgery were severely defective in salt chemotaxis learning, suggesting that the AIA neurons are important regulators of salt chemotaxis learning. From these data we propose a model in which the PI 3 kinase pathway in the salt-sensing ASER neuron is activated by the insulin-like molecule INS-1 secreted from the AIA interneurons, and regulates salt chemotaxis learning. We thank Cathy Wolkow for the age-1 cDNA and Gary Ruvkun for the ins-1(nr2091) strain. (1) Saeki et al. 2001 J. Exp. Biol. 204, 1757-1764 (2) Tomioka et al. 2003 IWM