Kariya, M.J. et al. (2019) International Worm Meeting "Sulfolipids from P. pacificus."

Bose, N., Kariya, M.J.

[International Worm Meeting, 2019]

From tRNA-derived ascarosides to xylose based nucleosides, Pristionchus pacificus has been shown to make a large diversity of molecules. Through an activity-guided search for an alert pheromone, we uncovered a collection of novel sulfolipids from the exo-metabolome of P. pacificus. Using targeted fragment traces in HPLC-MSMS, we have uncovered a library of sulfolipids from the P. pacificus metabolome, including unprecedented cyclic sulfates, the first of their kind in nature.

Burdine RD et al. (1996) East Coast Worm Meeting "A C. ELEGANS FGF-LIKE MOLECULE IS INVOLVED IN SEX MYOBLAST MIGRATION."

Stern MJ, Burdine RD

[East Coast Worm Meeting, 1996]

The sex myoblasts (SMs) undergo an anterior migration in hermaphrodite larvae to final positions that flank the precise center of the developing gonad. There they divide to give rise to the sex muscles necessary for egg laying. Mutations in egl-15 or egl-17 interfere with proper SM migration and confer posteriorly displaced SMs and an egg laying defective phenotype (Egl). egl-15 has been cloned previously and shown to encode a fibroblast growth factor receptor (FGF-R) homologue (1). We have characterized and cloned egl-17 in order to understand its role in SM migration. The genomic region containing egl-17 was identified and subsequently narrowed to 7 kb based on germline transformation rescue of the Egl defect of egl-17 mutants. Primers to two regions shown to be important for egl-17 rescue activity were used to obtain the cDNA by RT-PCR. The egl-17 cDNA encodes a product with significant homology to the fibroblast growth factor (FGF) family of proteins and represents one of the first functional invertebrate FGFs known. All ten known mutant alleles of egl-17 are viable as homozygotes and have similar distributions of displaced SMs. Analysis of the molecular lesions associated with egl-17 in these alleles has shown that all have the potential to eliminate egl-17 function; therefore, the SM migration defect represents complete loss of egl-17 function. We propose that EGL-17 may act as a ligand for EGL-15 (FGF-R). Besides affecting SM migration, mutations in egl-15 can result in larval arrest, scrawny body morphology, and the ability to suppress the effects of mutations in clr-1(1). Since elimination of egl-17 function only results in SM migration defects, EGL-17 is likely to be an SM migration-specific ligand and additional ligands presumably play a role in the other functions of EGL-15. 1. DeVore, D.L., Horvitz, H.R. and Stern, M.J. Cell 83, 611-620 (1995)

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

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

[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.

Tinya Fleming et al. (2003) International Worm Meeting "The FGF homolog EGL-17 acts with its receptor EGL-15 to repel migrating CANs"

Tinya Fleming, Fred Wolf, Gian Garriga

[International Worm Meeting, 2003]

Fibroblast Growth Factors (FGF) and their receptors (FGFR) are required for cell migrations in many organisms. In C. elegans the FGF homolog EGL-17 acts through its receptor EGL-15 to attract the migrating sex myoblasts (SMs)1. We have found that egl-17 and egl-15 also play a role in the migrations of the CANs (canal-associated neurons), a pair of bilaterally symmetric neurons that migrate posteriorly during embryogenesis.Although egl-17 and egl-15 mutations do not display striking CAN migration defects on their own, they significantly enhanced the CAN migration defects seen in vab-8 and unc-34 mutants. An egl-17 translational fusion to GFP expressed in only two anterior head cells during the time of CAN migration, raising the possibility that EGL-17 repels the CANs posteriorly. To test this possibility we expressed egl-17 ectopically. Ectopic expression of egl-17 in the head rescued the CAN migration defects of vab-8; egl-17 and unc-34; egl-17 double mutants, while ectopic expression of egl-17 globally or posteriorly enhanced the defects of vab-8 and unc-34 mutants. These results suggest that EGL-17 functions as a repellent in CAN migration. This model predicts that EGL-15 should function cell autonomously in the CANs. In support of this model, we found that a Pceh-10::egl-15 transgene, which is predicted to express in the CANs and only a few other neurons, partially rescued the defects of a vab-8; egl-15 double mutant. We are currently testing if unc-34; egl-15 double mutants are also rescued by the transgene.There are two isoforms of EGL-15, which have separate functions. The EGL-15(5A) isoform is required for SM migration and the EGL-15(5B) isoform is required for viability.2 Preliminary results suggest that both EGL-15 isoforms are involved in CAN migration. We are currently characterizing the roles of the EGL-15 isoforms in directing CAN migration.1Burdine, R.D. et al. (1998) Development. 125, 1083-1093; 2S.J. Goodman and M.J. Stern (personal comm.)

Watanabe, Shigeki et al. (2011) International Worm Meeting "Three-dimensional molecular architecture of a cell using photo-activated localization microscopy and electron microscopy."

Chen, Pin-An, Richards, Jackson, Watanabe, Shigeki, Jorgensen, Erik, Frokjaer-Jensen, Christian, Hollopeter, Gunther, Hobson, Robert

[International Worm Meeting, 2011]

Mapping the molecular architecture of proteins within a cell is essential for understanding their function. Fluorescence microscopy has been widely used for this purpose. However, the diffraction limit of light limits this approach, since fluorophores that are within ~200 nm of each other cannot be resolved. Several microscopy techniques have been developed to break this limit, including photo-activated localization microscopy (PALM)[1] and related techniques (STORM[2] and fPALM[3]). PALM allows one to precisely pinpoint the location of a single molecule in a cell to within 10 nm. However, precise localization in a field of black is not useful. Where is that protein in the context of cellular structure? PALM images need to be overlaid on the structures visualized by electron microscopy. Therefore, a method to preserve fluorophores in tissues fixed and embedded in plastic needs to be developed. To preserve fluorescence in plastic, three conditions are required: hydration, limited oxidation from fixatives, and neutral pH. We found that use of 5% water, a less oxidative fixative (KMnO4), and hydrophilic plastic (GMA - pH8), were sufficient to preserve signals and morphology. To test the feasibility of fEM using PALM, we tagged histone, TOM-20, and liprin, with photo-convertible fluorescent proteins. We showed that histone, TOM-20, and liprin were successfully localized to the expected organelles - nucleus, mitochondrial membrane, and dense projection, respectively[4]. We also have mapped the a2d subunits of voltage-gated calcium channels in the nerve ring of C. elegans. We found the subunits to be exclusively localized to dense projections, supporting the previous localization pattern by immuno-EM[5]. This result suggests that the fEM is feasible for protein localization, and many proteins can be localized to their cellular structures if they can be tagged by fluorophores. References [1]Betzig, E. et al. Science 313, 1642-1645 (2006). [2]Rust, M.J., Bates, M. & Zhuang, X. Nat. Methods 3, 793-795 (2006). [3]Hess, S.T., Girirajan, T.P.K. & Mason, M.D. Biophys. J 91, 4258-4272 (2006). [4]Watanabe, S. et al. Nat. Methods 8, 80-84 (2011). [5]Gracheva, E.O., Hadwiger, G., Nonet, M.L. & Richmond, J.E. Neurosci. Lett 444, 137-142 (2008).

Chute, Christopher et al. (2015) International Worm Meeting "Neural circuits involved in octopamine succinylated ascaroside #9 mediated avoidance responses."

Srinivasan, Jagan, Chute, Christopher

[International Worm Meeting, 2015]

Innate behaviors are mediated by the integration of external stimuli and internal signals. These cues often take the form of small biogenic molecules, and range from exogenous compounds to endogenous neuromodulators. These molecules are known to govern behavior across all domains of life. Although the basic components have been characterized in complex systems, the underlying physiological and neural mechanisms are largely unknown. C. elegans is an ideal organism for studying the relationship between small molecules and behavior due to its ability to survive in an unpredictable habitat. In order to successfully navigate their environment, these nematodes must sense and respond to multiple external cues, including physiological state-dependent signals produced and secreted by conspecifics that communicate important ecological contexts. A novel compound of interest, produced exclusively by starved first larval stage worms, is the neurotransmitter-derived metabolite octopamine-succinylated ascaroside #9 (osas#9) [1]. This molecule elicits an acute avoidance response in conspecifics [1], suggesting the communication of unfavorable environmental conditions from starved L1 worms. The avoidance response can be quantified by observing reversal in the worm's locomotion upon application of a drop of osas#9. A reverse genetic approach revealed the involvement of the bioamine neurotransmitters glutamate, octopamine, and tyramine in the avoidance response. Furthermore, through neuronal ablations, the glutamanergic neuron ASH has been identified as the primary sensory neuron necessary for mediating response to this metabolite. Notably, assaying biosynthetic enzymes tdc-1 and tbh-1, known to be required for tyramine and octopamine biosynthesis [2], indicates that the absence of both neurotransmitters does not display a behavioral defect, but the absence of only the downstream neurotransmitter, octopamine, does. This implies the existence of either an uncharacterized antagonistic relationship between octopamine and tyramine, or of an uncharacterized biosynthetic salvage pathway. Since tyramine and octopamine are structurally similar to human catecholamines, understanding their role in governing fear-like states in C. elegans will provide insights on the role of bioamines in mediating human behavior.1. Artyukhin, A.B., et al. J Biol Chem, 2013. 288(26): p. 18778-83.2. Alkema, M.J., et al. Neuron, 2005. 46(2): p. 247-60.

Roy, V. et al. (2019) International Worm Meeting "DAF-18/PTEN links germline stem cell proliferation to oocyte needs by preventing the ovulation of unfertilized oocytes."

Roy, V., Gagne, O., Narbonne, P.

[International Worm Meeting, 2019]

The rapid germline stem cell (GSC) proliferation rates of well-fed young wild-type hermaphrodites require the activation of both insulin/IGF-1 and ERK/MAPK signalling. Inhibition of either pathway slows down GSC proliferation, while their simultaneous inhibition completely blocks it (Narbonne et al. 2017). We are beginning to understand how insulin/IGF-1 stimulates GSC proliferation, but we do not know how this process is influenced by MPK-1/MAPK activity. In an attempt to identify the ligand upstream of MPK-1 that may be responsible for promoting GSC proliferation, we used RNAi to inactivate known upstream MPK-1 regulators. We found that lin-3(RNAi) dramatically inhibited GSC proliferation in adult hermaphrodites. GSCs in lin-3(n1058) and let-23(sy1) mutants also display reduced proliferation. LIN-3 and LET-23 are however required to induce spermathecal dilation and ovulation in hermaphrodites (Clandinin et al. 1998). As oocyte accumulation inhibits GSC proliferation, we wondered whether LIN-3 was required to promote GSC proliferation only in the presence of oocytes. Consistent with this hypothesis, GSC proliferation was found to be normal in lin-3(n1058) mutants that had not yet produced oocytes, at the L4 stage. We also had found that unfertilized oocytes spontaneously activated and were ovulated in the absence of sperm in daf-18/PTEN mutants, preventing the proper accumulation of unfertilized oocytes and the inhibition of GSC proliferation (Narbonne et al. 2017). This defect is fully rescued by daf-18(+) re-expression specifically in the proximal somatic gonad, as driven by the fos-1a promoter. daf-18 encodes a PIP3 phosphatase. Incidentally, the activation of LET-23 by LIN-3 is thought to induce spermathecal dilation by raising IP3 levels, which induces the ITR-1 receptor to release Ca++. PLC-1 is a phosphatase that converts PIP3 to IP3 and is also required for ovulation (Kariya et al. 2004). We thus propose that, in the absence of DAF-18, spermathecal PIP3 levels are abnormally high, resulting in increased IP3 levels, causing LIN-3/LET-23-independent spermathecal dilation and ovulation. Consistent with this hypothesis, we found that daf-18(-) significantly restores the proliferation of GSCs in lin-3(n1058) mutant adults. Thus, our data together suggest that DAF-18 acts in the spermathecal sheath cells to link germline stem cell proliferation to oocyte needs, by preventing the ovulation of unfertilized oocytes in the absence of induction by LIN-3/LET-23.

Matthew K Robinson et al. (2001) International C. elegans Meeting "sem-3 encodes a cis-prenyltransferase homolog, an enzyme required for production of an essential lipid."

Matthew K Robinson, Michael J Stern

[International C. elegans Meeting, 2001]

In C. elegans , the egg-laying muscles are descended from a pair of M-derived cells known as the sex myoblasts (SMs). The SMs are born in the posterior of the animal during the L2 stage and migrate anteriorly, in response to multiple cues, to flank the center of the developing gonad.1 The development of functional egg-laying muscles requires their proper differentiation, attachment, and innervation in addition to the proper migration of the SMs. sem-3(n1655) was identified as a spontaneous Egl mutation with defects in SM migration and vulval muscle attachment. The final positions of the SMs in sem-3(n1655) hermaphrodites lack the precision found in wild type, but are very close to normal. The sex muscles derived from these SMs are normal both in number and in their expression of appropriate reporter constructs, but often fail to attach properly. The subtle nature of the SM migration defect makes it likely that the vulval muscle attachment defects are the cause of the Egl phenotype of sem-3(n1655) mutants. sem-3 was mapped to linkage group IV and found to be allelic to let-654. Standard germline transformation rescue of the Let and Egl phenotypes was used to identify the sem-3 open reading frame from among cosmids in the region; the presence of lesions in multiple sem-3 alleles confirmed the identification of the sem-3 gene. By sequence analysis, sem-3 encodes a member of the cis-prenyltransferase class of enzymes. This class of enzymes is responsible for the production of dolichol, a lipid molecule with essential functions in N-linked glycosylation, formation of GPI anchors, and regulation of membrane fluidity.2 BLAST analysis shows that SEM-3 is the only member of this class of enzymes in C. elegans and suggests that the lethality associated with sem-3 mutations may be due to a lack of dolichol. Consistent with an essential role for SEM-3 in a ubiquitous process such as N-linked glycosylation a sem-3 ::GFP reporter construct is expressed in a wide variety of tissues throughout development. Models for SEM-3 function in vulval muscle attachment will be discussed. 1. Chen, E.B. and Stern, M.J. (1998) TIGS 14, 322-327 2. Chojnacki, T. and Dallner, G. (1988) Biochem. J. 251, 1-9

Robinson MK et al. (2000) East Coast Worm Meeting "sem-3 encodes a cis-prenyltransferase homolog, an enzyme required for production of an essential lipid"

Stern MJ, Robinson MK

[East Coast Worm Meeting, 2000]

In C. elegans, the egg-laying muscles are descended from a pair of M-derived cells known as the sex myoblasts (SMs). The SMs are born in the posterior of the animal during the L2 stage and migrate anteriorly, in response to multiple cues, to flank the center of the developing gonad.1 The development of functional egg-laying muscles requires their proper differentiation, attachment, and innervation in addition to the proper migration of the SMs. sem-3(n1655) was identified as a spontaneous Egl mutation with defects in SM migration and vulval muscle attachment. The final positions of the SMs in sem-3(n1655) hermaphrodites lack the precision found in wild type, but are very close to normal. The sex muscles derived from these SMs are normal both in number and in their expression of appropriate reporter constructs, but often fail to attach properly. The subtle nature of the SM migration defect makes it likely that the vulval muscle attachment defects are the cause of the Egl phenotype of sem-3(n1655) mutants. sem-3 was mapped to linkage group IV and found to be allelic to let-654. Standard germline transformation rescue of the Let and Egl phenotypes was used to identify the sem-3 open reading frame from among cosmids in the region; the presence of lesions in multiple sem-3 alleles confirmed the identification of the sem-3 gene. By sequence analysis, sem-3 encodes a member of the cis-prenyltransferase class of enzymes. This class of enzymes is responsible for the production of dolichol, a lipid molecule with essential functions in N-linked glycosylation, formation of GPI anchors, and regulation of membrane fluidity.2 BLAST analysis shows that SEM-3 is the only member of this class of enzymes in C. elegans and suggests that the lethality associated with sem-3 mutations may be due to a lack of dolichol. The bases for the vulval muscle attachment defects, as well as the subtle SM positioning defects, in sem-3(n1655) animals are not currently known. Experiments to address the role of SEM-3 in vulval muscle attachment, as well as the attachment of other muscles, will be discussed. 1. Chen, E.B. and Stern, M.J. (1998) TIGS 14, 322-327 2. Chojnacki, T. and Dallner, G. (1988) Biochem. J. 251, 1-9

Brightbill D et al. (1998) East Coast Worm Meeting "DELETION OF AN X CHROMOSOME REGION THAT INCLUDES A RalGDS ENHANCES ras AND mig-2 GAIN-OF-FUNCTION MUTATIONS"

Klampert K, Brightbill D, Wightman BC

[East Coast Worm Meeting, 1998]

gm27 is a deletion that removes approximately nine predicted genes on the left arm of the X chromosome. One of these genes is the nuclear receptor fax-1 (see abstracts by Carmean et al. and Wightman et al.). gm27 homozygous animals are phenotypically very similar to animals that are homozygous for a point mutation in the fax-1 gene (gm83). gm27 homozygotes do not appear more severely affected than fax-1(gm83) homozygotes, other than a reduction in brood size. This suggests that deletion of the genes flanking fax-1 results in a relatively subtle effect on the animal. Among the flanking genes that are deleted by gm27 is one that encodes a predicted RalGDS (F28B4.2), a guanine nucleotide-exchange factor for Ral G proteins. Biochemical studies in vertebrates and C. elegans (K. Kariya, personal communication) have demonstrated that RalGDS binds to activated ras protein. Therefore, RalGDS has been proposed to be a mediator of ras signaling. gm27 homozygotes do not display defects in vulval or male tail development, indicating that deletion of this RalGDS alone does not affect ras signaling significantly. We considered the possibility that the F28B4.2 RalGDS could have a modulatory effect on ras signaling. We created gm27; ras(gf) double mutants to determine if deletion of RalGDS enhances or suppresses ras mutations. gm27; ras(gf) double mutants displayed a fully-penetrant Muv defect, unlike ras(gf) alone. Moreover, approximately 20% of double mutants arrested during late larval or early adult stages and displayed a clear phenotype. These phenotypes are similar to those observed when activated ras is overexpressed. Therefore, deletion of this region of the X chromosome appears to enhance activated ras. fax-1(gm83); ras(gf) double mutants are similar to ras(gf) alone, indicating the enhancement of activated ras is not due to deletion of fax-1. We are currently testing whether this enhancement is dependent on deletion of RalGDS, another gene, or a combination of genes. If the enhancement is due to deletion of RalGDS, this would suggest that, counter to the commonly-accepted model, RalGDS functions as a negative modulator of ras signaling. We also tested the possibility that deletion of RalGDS could have effects on mutations in genes that function in other signal transduction pathways. The gm27 deletion did not obviously enhance mig-2(gm103) or unc-73 (e936). However, gm27 mig-2(gm38) double mutants displayed an increase in the penetrance of the withered-tail phenotype associated with mig-2 and significant lethality. Therefore, gm27 may also enhance gain-of-function mutations that perturb Rho family signaling. This raises the possibility that the F28B4.2 RalGDS may be a negative modulator of two signal transduction pathways.