Thomas Hills et al. (2001) International C. elegans Meeting "Genetic analysis of the neuromodulatory control of search behavior in C. elegans"

Andres Villu Maricq, Thomas Hills, Fred Adler

[International C. elegans Meeting, 2001]

Following removal from food, many animals engage in an area-restricted search, a stereotypic search strategy characterized by a concentrated local search followed by a more directed global search. To understand the neuromodulatory control of the plasticity underlying area-restricted search behavior, we have quantified this search phenotype in C. elegans . Using a computer-assisted motion analysis system, search paths for a small population of worms can be analyzed for dynamic changes in turning angle, speed, and other statistical properties. The sensitivity of this system to subtle behavioral changes allows for the general construction of a library of quantified phenotypic effects of genetic mutations on locomotion. Our preliminary results suggest that the area-restricted search behavior is generated by a two-state neural circuit controlling forward and backward motion that is tuned by dopamine via a possible kinetic modulation of glutamate receptors. A separate but not independent pathway controls locomotory rate and appears to be mediated by serotonin and glutamate. We are currently engineering worms with dopaminergic neurons that are either prematurely apoptotic, constitutively depolarized, or constitutively hyperpolarized. By crossing these worms into various genetic backgrounds and using specific pharmacological agents, we will test the hypotheses that dopamine and serotonin act presynaptically to glutamate and that serotonin inhibits the effects of dopamine in the generation of this ecologically ubiquitous search behavior.

Thomas T Hills et al. (2002) West Coast Worm Meeting "Genetic Analysis of the Neuromodulatory Control of Search Behavior in C. elegans"

Thomas T Hills, Andres Villu Maricq, Fred Adler

[West Coast Worm Meeting, 2002]

Immediately following removal from food, C. elegans engages in area-restricted search, a behavior found in a wide variety of animals. In C. elegans, this behavior is characterized by an initial high frequency of sharp turns that then decays to less frequent turning over time. To understand the neuromodulatory control of search behavior in C. elegans, we used a computer assisted path analysis system (DIAS) to quantify the temporal dynamics of worm movement under genetic, neural, and pharmacological manipulations. Our results show that C. elegans hermaphrodites perform an area-restricted search after removal from or following brief encounters with food. Glutamate receptor function in the command interneurons, known to control reversal frequency, is required for this behavior. Mutations that modify glutamate-gated currents also modify turning frequency. The dopaminergic neurons in C. elegans are presynaptic to the glutamatergic command interneurons, suggesting a possible role for dopamine in the modulation of glutamatergic signaling. Exogenous application of dopamine increases the frequency of high angled turns, whereas dopamine antagonists inhibit this effect. Genetic ablation of the dopaminergic neurons eliminates area-restricted search. We also show that normal glutamate activity is required for dopaminergic modulation of behavior. Taken with previous results from other labs, our work suggests that dopaminergic neurons respond to food by modulating glutamatergic signaling. This increases the frequency of high angled turns and generates an area-restricted search following removal from food.

Saleel Raut et al. (2008) C.elegans Neuronal Development Meeting "HLH-3, a C. elegans Achaete/Scute protein, has a role in the axonal pathfinding of hermaphrodite-specific motor neuron (HSN) that is independent of UNC-40."

Aixa Alfonso, Saleel Raut

[C.elegans Neuronal Development Meeting, 2008]

The basic helix loop helix protein, HLH-3, is a member of the achaete/scute family in C. elegans hlh-3 mutants have an egg-laying defective (Egl) phenotype. We have shown (Doonan et al., 2008 submitted) that the mutant phenotype results from abnormal axonal pathfinding in hermaphrodite-specific motor neurons (HSNs) that fail to innervate the vulval muscles. The NETRIN/UNC-6 receptor UNC-40/DCC is necessary for the guidance of the HSN axons (Garriga et al.,1993; Chan et al., 1996; Gitai et al., 2003; Adler et al., 2006). We hypothesized that mutant animals are not uncoordinated (Unc). Currently we are looking at SYG-1, a receptor on HSN''s that binds with the guidepost signal SYG-2 for proper synapse formation (Shen et al., 2004), as a possible target of HLH-3.

Ward S et al. (1983) Worm Breeder's Gazette "Monoclonal and Polyclonal Antibodies that Bind to Sperm Antigens"

Hogan E, Ward S

[Worm Breeder's Gazette, 1983]

We have examined a number of monoclonal and polyclonal antisera generously provided to us by other labs in order to assess their binding to sperm antigens. The antisera include the following: two rabbit sera and eight mouse monoclonals raised against muscle proteins by Ross Francis: five anti-sperm mouse monoclonals prepared by Susi Strome and obtained initially from Michael Klass; eight anti-sperm mouse monoclonals prepared by Fred Pavalko and Tom Roberts; and a rabbit anti-glyceraldehyde phosphate dehydrogenase (GAPDH) serum obtained from Patrice Yarborough and Ralph Hecht. Our interest in these sera is to use them to identify new sperm antigens whose cellular localization and developmental history can be compared with the sperm specific antigens we have alrealy examined. All the antisera were first assayed for tissue specificity by a solid phase immunoassay with proteins from sperm, eggs, larvae or fem- 1( hc17) hemaphrodites which have no sperm. Those antisera that showed significant binding to sperm proteins were then characterized further by immunostaining of sperm or fem-1 proteins transferred from 1-D or 2- D gels to nitrocellulose ('Western transfers'). In addition the cellular localization of antigens was determined by immunofluorescence.

McNeil K et al. (1984) Worm Breeder's Gazette "mitochondrial DNA or how to date a worm."

McNeil K, Rose AM

[Worm Breeder's Gazette, 1984]

A strain of C. isolated from the Kitsilano district of Vancouver by our colleague Fred Dill. Kitsilano derives its name from the North American Indian tribe. This strain is a self- breeding hermaphrodite and when N2 males are crossed with Kitsilano hermaphrodites, fertile males and hermaphrodites are produced. When grown on Petri plates in the laboratory, Kitsilano individuals have an 'unhealthy' appearance and a longer developmental time than N2. This strain produces a large number of morphological variants (paralyzed and dumpy worms) that do not breed true. After treatment with 0.01 M EMS, an F2 individual was recovered which has an N2-like appearance and growth rate. Thus we have two Kitsilano lines, the original and the modified (Kits M). On genomic blot-hybridizations, Kitsilano has an N2-like Tc1 pattern with a couple of altered Tc1 fragments. We have now prepared mitochrondria from the N2 strain, and are in the process of comparing the mitochrondrial DNA restriction patterns of the N2, BO and Kits strains. We hope to be able to estimate the time of evolutionary divergence of C. elegans strains. This may tell us if Kitsilano arrived in Vancouver in 1973 (with David Baillie from Cambridge) or is a truly Canadian strain of C. elegans (eh?).

Sawa H et al. (1996) East Coast Worm Meeting "Molecular and genetic analyses of lin-17, which encodes a putative receptor required for asymmetric cell divisions"

Horvitz HR, Sawa H

[East Coast Worm Meeting, 1996]

Asymmetric cell division is a fundamental mechanism responsible for increasing cellular diversity during development. lin-17 is required for the asymmetric divisions of a number of cells in C. elegans (1). lin-17 encodes a putative seven-transmembrane protein similar to the frizzled gene product of Drosophila. frizzled is required for the correct polarities of a number of cells and tissues (2). Using a reporter construct, we have found that lin-17 is expressed in cells prior to their asymmetric cell divisions and also in both daughter cells after the divisions. Our results suggest that lin-17 encodes a receptor that regulates the polarities of cells undergoing asymmetric cell divisions. We have identified the molecular lesions of twelve lin-17 alleles. One, n3091, has a stop codon close to the N-terminus of the coding sequence, suggesting that it is a null allele. n669 causes a Gly-to-Arg change in the middle of the seventh transmembrane domain. rh71 causes a Gly-to-Asp change in the third transmembrane domain close to a presumptive cytoplasmic region. The residues affected in these mutants may define interaction sites of the LIN-17 receptor with its ligands or effector molecules. We are screening for suppressors of these missense mutations in an attempt to identify genes in the lin-17 signaling pathway. (1) Sternberg, P.W. & Horvitz, H.R. Dev. Biol. 130, 67-73 (1988). (2) Adler, P.N. BioEssays 14, 735-741 (1992).

Wu JC et al. (2007) Mol Biol Cell "PAR-3 and PAR-1 inhibit LET-99 localization to generate a cortical band important for spindle positioning in Caenorhabditis elegans embryos."

Wu JC, Rose LS

[Mol Biol Cell, 2007]

Monitoring Editor: Fred Chang The conserved PAR proteins are localized in asymmetric cortical domains and are required for the polarized localization of cell fate determinants in many organisms. In C. elegans embryos, LET-99 and G protein signaling act downstream of the PARs to regulate spindle positioning and ensure asymmetric division. PAR-3 and PAR-2 localize LET-99 to a posterior cortical band through an unknown mechanism. Here we report that LET-99 asymmetry depends on cortically localized PAR-1 and PAR-4 but not on cytoplasmic polarity effectors. In par-1 and par-4 embryos, LET-99 accumulates at the entire posterior cortex, but remains at low levels at the anterior cortex occupied by PAR-3. Further, PAR-3 and PAR-1 have graded cortical distributions with the highest levels at the anterior and posterior poles respectively, and the lowest levels of these proteins correlates with high LET-99 accumulation. These results suggest that PAR-3 and PAR-1 inhibit the localization of LET-99 to generate a band pattern. In addition, PAR-1 kinase activity is required for the inhibition of LET-99 localization and PAR-1 associates with LET-99. Finally, examination of par-1 embryos suggests that the banded pattern of LET-99 is critical for normal posterior spindle displacement and to prevent spindle misorientation caused by cell shape constraints.

Meenakshi Balaraman et al. (2008) C.elegans Neuronal Development Meeting "hlh-3has a role in regulating unc-40 expression in the VC neurons of C. elegans"

Aixa Alfonso, Saleel Raut, Meenakshi Balaraman

[C.elegans Neuronal Development Meeting, 2008]

HLH-3, a basic helix-loop- helix (bHLH) transcription factor in C. elegans has been shown to be required for differentiation of HSN neurons. (Doonan et al., 2008, submitted). Worms that lack HLH-3 function are egg-laying defective (Egl). The Egl phenotype is likely to be a result of abnormal differentiation and pathfinding defects of the HSNs. (Doonan, et al., 2008, submitted).Since work by many others (Garriga et al., 1993; Gitai et al., 2003;Adler et al., 2006; Chang et al., 2006) has shown that UNC-40/DCC mediates the reception to attractive cues (UNC-6/netrin) for ventral projection of the HSN axon, we asked whether hlh-3 mutants lack UNC-6 signaling in the HSNs. In order to test this, we characterized the expression of unc-40::gfp in an hlh-3 mutant background. Expression of this functional unc-40 reporter is variable in the HSNs (See Raut, et al., this meeting). However, to our surprise, the VC4 and VC5 neurons in the hlh-3 mutant lack the expression of unc 40::gfp. The absence of unc-40::gfp expression is not due to the absence of the VCs, as staining for UNC-17 (vesicular acetylcholine transporter) in the hlh-3 mutant, showed the presence of VC4 and VC5. We report on characterization of the VC axons in hlh-3 and unc-40 mutants and we are further interested in studying whether a defect in VC function is involved in the Egl phenotype of the hlh-3 mutant.

ZHANG, Gaotian et al. (2019) International Worm Meeting "A genetically divergent C. elegans wild isolate exhibits extremely high male frequency."

Andersen, Erik, Crombie, Tim, Zhang, Gaotian, Zdraljevic, Stefan

[International Worm Meeting, 2019]

Caenorhabditis elegans is an androdioecious nematode with hermaphrodites and males. A self-fertilizing hermaphrodite rarely produces males, with frequencies of approximately 0.1% in the laboratory strain N2. By contrast, a hermaphrodites fertilized by males have 50% male progeny. Many studies have suggested that self-fertilization is the primary reproduction mode of C. elegans in nature, and the transition from obligately outcrossing might be recent (Cutter et al., 2008). Natural variation of male frequencies were also observed in C. elegans, ranging from 0.1% to 35% (Teotonio et al., 2006; Anderson et al., 2010). In a recent sampling of Caenorhabditis in Hawaii (see abstract by Crombie et al.), we isolated a C. elegans strain, named ECA701, which exhibited extremely high male frequencies of 34% - 57% and low brood sizes of 80 - 170 by selfing of hermaphrodites. Mating of ECA701 between hermaphrodites and males showed male frequencies of 47% - 70% and brood sizes of 150 - 290. We further found that ECA701 produced embryos with high levels of embryonic lethality (30% in young adults), which is exacerbated in older adults (60% in two-days old adults). Investigations on meiotic prophase I demonstrated that ECA701 is defective in the meiotic processes (see abstract by Adler et al.). Notably, using whole genome sequencing and phylogenetic analysis, we also found that ECA701 is one of the most divergent C. elegans strains that have yet been described. Taken together, ECA701 provides a great opportunity to improve our understanding on the possible ancestral state and the evolution of reproduction mode in C. elegans.

CM Loer et al. (1999) International C. elegans Meeting "Continuing characterization of serotonergic marker genes and serotonin-deficient mutants"

GP Merritt, CM Loer

[International C. elegans Meeting, 1999]

We are interesting in the regulation of genes used by serotonergic neurons in C. elegans. We and others have identified several genes that are expressed or are likely to be expressed in serotonergic neurons, including genes encoding serotonin synthetic enzymes Tryptophan Hydroxylase (TrpH) and Aromatic L-Amino Acid Decarboxylase (AAADC), and the biopterin cofactor synthetic enzyme GTP Cyclohydrolase I (GCH). These genes may correspond to the mutationally-identified genes cod-5, bas-1, and cat-4, respectively. We have previously rescued the serotonin- and dopamine-deficient mutant bas-1 by injecting DNA containing two predicted AAADC genes (thanks to Fred Wolf, Garriga lab, for this clone). One or both of these ORFs may be needed for serotonin synthesis. We plan to test this by injecting clones mutated in each of the ORFs. The two predicted genes are separated by only 369 bp; we are also testing whether they are expressed together as an operon. The mutant cat-4 (e1141)V is serotonin- and dopamine-deficient and appears to have a leaky cuticle. We are currently comparing the cuticles of cat-4 mutants and wild type worms using EM to see whether any structural differences are apparent. Sequencing through the region in which cat-4 maps genetically identified a candidate gene with homology to GCH, which catalyzes the first step in biopterin cofactor synthesis. Biopterin is a cofactor used by all aromatic amino acid hydroxylases, which include tryptophan hydroxylase (serotonin synthesis), tyrosine hydroxylase (dopamine synthesis) and phenylalanine hydroxylase (tyrosine synthesis/phenylalanine catabolism). We are also injecting the F32G8 cosmid into cat-4 mutants to see whether the clone will rescue.