Morphew JJ et al. (1997) International C. elegans Meeting "MUTATION ACCUMULATION FOR CHEMOSENSORY BEHAVIOR"

Morphew JJ, Phillips PC

[International C. elegans Meeting, 1997]

In each generation the genetic variation within a population increases because of the accumulation of mutations and decreases in response to natural selection and genetic drift. Understanding the dynamics of this process is complicated by the fact that multiple genes may simultaneously affect the trait of interest. Mutations of small effect could accumulate at any or all of these loci, yielding a large phenotypic effect in the aggregate. C. elegans provides a useful model system for studying mutation accumulation because it is easy to propagate essentially clonal individuals using selfing, and it is possible to minimize environmental variation in a laboratory setting. In collaboration with Peter Keightley and Armando Caballero (Univ. Edinburgh), fifty isogenic lines that were all derived from the same inbred individual and kept separate via selfing where assayed for their chemotactic responses to benzaldehyde at 50 nl (an attractive concentration) and 500 nl (a repulsive concentration). Significant variation among the lines was observed after sixty generations of isolation, indicating an accumulation of mutational variation on the order of 10-3 to 10-4 of the environmental variation per generation. Mutational variation at 50 nl was an order of magnitude higher than that detected at 500 nl, with most of that difference apparently resulting from a mutation of large effect in one line. Results from this study are important to the understanding of the maintenance of variation for polygenic traits in natural populations, as well as consequences of finite population size in conservation genetics.

Freedman, Benjamin L et al. (2013) International Worm Meeting "Caenorhabditis-in-Drop (CiD) method to measure worm behavior and longevity."

Yuan, Jinzhou, Chuan, Han-Sheng, Norton, Michael, Raizen, David, Belfer, Samy, Freedman, Benjamin L, Bau, Haim

[International Worm Meeting, 2013]

Lethargus-a sleep-like stage occurring during C. elegans larval development-is regulated by some of the same mechanisms that regulate sleep in mammals and fruit flies, suggesting that lethargus can be used to study sleep. To monitor the behavioral quiescence of 24 nematodes simultaneously, we have developed the Caenorhabditis-in-Drop (CiD) method, which entails automated videography of worms housed individually in 125 nL aqueous droplets on a polydimethylsiloxane (PDMS) surface and covered with mineral oil. We used the CiD method to demonstrate reduced quiescence in kin-2 mutants, and to reproduce previous observations of increased quiescence in animals over-expressing LIN-3C (Van Buskirk and Sternberg 07). We found that egl-4 mutants, which have reduced quiescence when housed on an agar surface (Raizen et al, 08), had no change in lethargus quiescence in CiD. This indicates that the chamber used to house the animal can affect behavior and raises a note of caution in the interpretation of both positive and negative results. In comparison to behavior on an agar surface, animals in CiD were more active and showed reduced survival, with most animals dead after one week. Housing the worms in a 10-fold higher volume (1250 nL) of aqueous solution on a PDMS surface increased survival in comparison to survival in 125-nL droplets, indicating that reduced longevity is not explained by the liquid habitat, by exposure to mineral oil, or by exposure to the PDMS surface. Surprisingly, animals survived longer in 125-nL droplets that previously housed a worm for a week.

Kotaro Kimura et al. (2005) International Worm Meeting "Different types of plasticity of avoidance behaviors."

Isao Katsura, Kotaro Kimura

[International Worm Meeting, 2005]

Although C. elegans exhibits several types of behavioral plasticity (ex. Rankin, 2004 Curr. Biol., 14, R617), we know little about plasticity of its avoidance behaviors. Because avoidance cues are toxic and/or hazardous signals, we speculated that the animals may exhibit plasticity other than adaptation in avoidance behaviors. We studied changes in avoidance behaviors of N2 animals after persistent stimulation using higher osmolarity, 1-octanol or 2-nonanone, which are sensed mainly by different sensory neuron classes respectively. For osmolarity assay, we used quadrant plates to obtain temporally more stable stimulus than the traditional "ring" assay; For repellent odor assay, we used 9 cm round plates and scored the average relative distance of the animals from the odor source. Osmolarity: Avoidance behavior to higher osmolarity was suppressed after starvation as well as after pre-exposure to the same stimulus for 1 hr. This suppression after pre-exposure is likely due to the adaptation of ASH neuron (Hilliard et al., 2005 EMBO J., 24, 63). 1-octanol: After starvation, avoidance behavior to 1-octaol (6 - 600 nL) was also suppressed, as similarly reported by Chao et al. (2004 PNAS, 101, 15512). However, after animals had been pre-exposed to 90 nL of the same odor during starvation, the avoidance behavior was not suppressed, and its magnitude was similar to that of naive animals. This modulation of avoidance behavior by pre-exposure could be explained either by suppression of the starvation-induced suppression of avoidance behavior, or by enhancement of the suppressed avoidance behavior. 2-nonanone: In contrast to 1-octanol, avoidance behavior to 2-nonanone was not suppressed by starvation. Still, pre-exposure to 30 nL of 2-nonanone during starvation significantly enhanced the avoidance behavior: Magnitude of the avoidance behavior to 200 nL of 2-nonanone of the pre-exposed N2 animals was enhanced (p < 0.001) and comparable to that of the behavior to 600 nL of 2-nonanone of unexposed animals. This result suggests that the pre-exposure may enhance the sensitivity of the sensory neuron to 2-nonanone by about 3-fold. Alternatively, the enhancement could be due to modulation of the locomotion. The pre-exposure in the presence of food also enhanced the avoidance behavior although it was not statistically significant. We are interested in these pre-exposure-induced modulations of avoidance behaviors because the plasticity of avoidance behaviors may be much more complicated than we expected. We are currently studying the plasticity in the following aspects: (1) its specificity and persistence, (2) responsible sensory neurons, and (3) genes required for the phenomenon.

Hennan M et al. (1991) International C. elegans Meeting "STILL MORE ON lin44"

Horvitz HR, Hennan M

[International C. elegans Meeting, 1991]

At the last C. elegans meeting, we reported the isolation of the mutation nl 792, which causes certain asymmetric cell divisions to reverse polarity. We have continued our analysis of the role of the gene defined by this mutation, lin44 1. Genetic analysis. We have isolated a new lin-44 allele, n2111, in a non-complementation screen. Both nl 792 and n2111 behave similarly in homozygotes, deficiency heterozygotes, and nl 792/n2111 transheterozygotes. The two alleles differ in that nl 792 is suppressed by a single copy of the amber suppressor sup-7, while n2111 is not suppressed. These alleles could define the null phenotype of lin44. Cell-interactions. Mutations in lin44 reverse the polarity of cell divisions in the B, F, U, and T lineages. These blast cells lie in the posterior of the male and are related primarily by position rather than by lineal history, suggesting that cell-interactions might be involved in defining the polarities of asymmetric cell divisions in these lineages. Chisholm and Hodgkin (Genes Dev. 3: 1413, 1989) have shown that when the B cell is kined with a laser microbearn in wild- type males, the polarity of the F and U lineages are reversed as they are in lin44 mutant males. Thus, the B cell influences the polarities of the F and U cells. What cell or cells influence the polarities of B and T? To answer this question, we have killed the mother of the B cell in both wild-type and lin44 embryos and looked at the polarity of T. We have not observed an effect on the T cell lineage in either case, and conclude that B does not influence the polarity of T. In an analogous experiment, we have killed the T cells before B is born in the embryo and looked for an effect on the polarity of the B cell division. We have not observed an effect in wild-type animals. Results with lin44 animals are not yet conclusive, although it appears T does not influence the polarity of B. Molecular analysis. To learn how lin44 controls the polarity of asymmetric cell division at the molecular level, we are cloning the locus. We have mapped the endpoints of the small deficiency hDp (which spans lin44 ) to the unc-73 contig by hybridization of labeled cosmids to wildtype and deficiency genomic DNA. Microinjection of cosmids in this interval does not rescue lin44. However, part of the interval is represented only by YAC clones, and we suspect that lin44 lies in this 'YAC gap.' We have injected the 350 kb YAC clone Y48F5 into lin44 animals and obtained two rescued lines. We are currently attempting to nanow down the size of the rescuing DNA.

Gerami B et al. (1997) International C. elegans Meeting "MAPPING QUANTITATIVE TRAIT LOCI FOR CHEMOTAXIS: CORRESPONDENCE BETWEEN QTL AND odr CANDIDATE LOCI"

Phillips PC, Gerami B, Morphew JJ

[International C. elegans Meeting, 1997]

Understanding the genetics of complex traits is challenging because it can be difficult to separate out the effects of the numerous genes affecting the trait. C. elegans can serve as a useful model system for studying complex traits because of the wealth of genetic and functional information available. Chemotaxis provides an ideal trait for this type of study because it ecologically important to the nematodes, as well as likely to be influenced by many genes (a number of which are known). In collaboration with Thomas Johnson and David Shook [1] and Robert Shmookler Reis and Robert Ebert [2], who provided genotyped lines generated in a cross between N2 and BO, we have assayed the chemotaxic response of 180 recombinant inbred lines to benzaldehyde at two concentrations. These concentrations, 50 nl and 500 nl, respectively lead to an attraction or repulsion response. Separate analysis of the lines from the two labs yield different results. The SR lines show a QTL mapping to the fourth chromosome, whereas the TJ lines show several QTL, primarily on the X chromosome. The QTL on X map to the same locations as two mutants known to block the response to benzaldehyde, odr-1 and odr-5 [3]. We are in the process of evaluating these candidate loci as the actual QTL using backcrossing and complementation testing approaches. Our hope is to create a model system for studying a variety of quantitative genetic questions. [1] Genetics 142:801 (1996), [2] Genetics 135:1003 (1993), [3] Bargmann et al., Cell 74:515 (1993)

Katz WS et al. (1991) International C. elegans Meeting "POLARITY IN THE VULVAL LINEAGES."

Katz WS, Sternberg PW

[International C. elegans Meeting, 1991]

The 2 vulval precursor cells (VPCs), PS.p and P7.p, produce asymmetric lineages of mirror-image symmetry around the 1 lineage. This fixed polarity is apparent in the orientation of the final nuclear divisions and migrations, which produce characteristic arrangements of daughter nuclei. To learn how signals from other cells might influence the polarity of the 2 lineages, we use lin-12(nl37) animals, in which all six VPCs form 2 lineages. This allows us to examine polarity at a range of positions. Because the VPCs in nl 3 7 animals proliferate whether or not inductive signal from the gonad is present, we have performed cell ablation experiments to examine the role of the gonad, and of neighboring VPCs, in specifying 2 lineage polarity. By Nomarski observation of intact nl 37 animals, P(3S).p Iineages typically are oriented to the posterior, P6.p to the anterior, and the polarities of P7.p and P8.p are variable. In the absence of the entire gonad all six lineages have a posterior orientation. Therefore, the gonad influences the polarity of the P(6-8).p lineages. One model is that all of the lineages have a posterior-oriented default polarity, which can be reversed in the P(6-8).p lineages in response to a signal from the gonad. This is unlikely to be the same signal that induces VPC proliferation: n 13 7 animals lack an anchor cell, which is required for vulval induction in wild type animals. Furthermore, lineage orientation appears to be independent of the vulval induction pathway since the polarity of P(6-8).p can be anterior in nl 37;Vul double mutants. We plan funher ablations to identify the gonadal cells needed for anterior-oriented lineages, (and to examine whether VPC-VPC interactions also play a role in orienting lineage pol arity ) . Mutations in the lin-18 gene reverse or abolish the polarity of the 2 lineages; some animals have normal 2 lineages. Since the two existing lin-l 8 alleles are incompletely penetrant, we have isolated a deficiency covering the lin-l 8 locus, syDfl, to help define the null phenotype. Either lin-l 8 allele over syDf l gives an increased percentage of 'bivulva' animals (resulting from a reversed P7.p Iineage), though penetrance is still incomplete. Since these alleles are viable over a deficiency, we are screening for null alleles by an Fl noncomplementation screen. As a first step to cloning this locus, we are analyzing congenic strains to identify TcI polymorphisms linked to lin-l 8.

Lambie EJ et al. (1991) International C. elegans Meeting "q425: A STERILE VUL, BUT STILL INTERESTING."

Lambie EJ, Kimble JE

[International C. elegans Meeting, 1991]

During an F2 screen for steriles, we isolated a recessive mutation ( q425 I) that produces an interesting combination of phenotypes. Homozygous q425 hermaphrodites are both sterile and vulvaless. Males are superficially normal, but they have not been tested for mating ability. Both males and hermaphrodites are capable of producing sperm; however, hermaphrodites never produce oocytes. In adult hermaphrodites, the proximal pachytene gerrnline nuclei tend to accumulate in the loop region of the gonad, instead of resuming meiosis and proceeding around the loop. One interpretation of this phenotype is that proximal pachytene nuclei normally receive a signal that induces them to resume meiosis and proceed around the loop into the ventral ovary. Some aspect of this signalling process could be disrupted by q425. To learn more about the possible influence of q425 on intercellular signalling, we examined its effect on vulval induction. Inspection of L3s revealed that q425 homozygotes possess an anchor cell, but the vulval precursor cells appear to divide only once, and then enter the hypodermal syncytium. This phenotype is similar to that of some other vulvaless mutants, let-23, lin-2, lin-3, lin-7, and lin-10 (Ferguson, Sternberg and Horvitz, 1987). q425 also resembles this group of vulvaless mutations in that it is not epistatic to a lin-12(d) mutation: q425; lin-12(nl37d) double mutants are Muv (and sterile). This indicates that q425 does not prevent vulval lineages per se, but may affect the decision to adopt a vulval fate. To further subclassify q425, we examined its epistatic relationship to the synMuv mutations, lin-8(nl l l ) and lin-9(nl l 2). q425; lin-8; lin-9 triple mutants are sterile and vulvaless. Mutations in lin-2, lin-7 and lin-10 are also epistatic to synMuv mutations, but lin-3 mutations are not. Because the multivulva phenotype produced by the synMuv mutations does not depend on the presence of the anchor cell, it is thought that lin- 2, lin-7 and lin-10 (and probably q425) are required for the response to the anchor cell signal, whereas lin-3 is required for its generation. q425 maps to the left arm of chromosome I, nearfog-l. No other vulvaless mutations have been reported for this region, so it is possible that q425 represents a newly identified gene. The pleiotropic effects of q425 suggest that it may disrupt cell-cell interactions in many tissues. In this regard, it is notable that while q425 single mutants are fairly well coordinated, q425 dpy-S(e61) double mutants exhibit an unusual shimmying behavior when induced to move backwards. The basis for this coordination defect is not known, but could involve neuraVmuscular signal transduction or the establishment of proper neuronal connectivity. Experiments are in progress to learn more about the genetics and phenotype of q425. (E.J.L. is supported by Damon Runyon-Walter Winchell postdoctoral fellowship DRG-989.)

Basson M et al. (1991) International C. elegans Meeting "MUTANTS DEFECTIVE IN HSN DIFFERENTIATION."

Horvitz HR, Basson M

[International C. elegans Meeting, 1991]

The HSNs, a pair of serotoninergic neurons, innervate the vulval muscles and drive egg-laying. The HSNs are born and migrate during embryogenesis, but do not differentiate into functional neurons until adulthood. The differentiated phenotypes of the HSNs that we monitor are (l) a change in morphology as visualized with Nomarski optics, termed hood formation, at approximately the time of the L3 molt; (2) axonal outgrowth; and (3) serotonin expression. We hope to understand how the timing of HSN differentiation is controlled and how the decision to differentiate leads to the expression of the differentiated phenotype. Mutations in several genes disrupt normal HSN differentation. Precocious or retarded mutations in the heterochronic genes cause the precocious or retarded onset of HSN differentiation (G. Garriga, WBG 11(2):101,1990). Furthermore, mutations in three other genes, unc-86, sem4 and egl45, block the expression of all three differentiated phenotypes of the HSNs. It seems likely that unc-86, which encodes a putative transcription factor, functions directly in regulating the expression of genes required for differentiation, since the temperature-sensitive period for unc-86 in controlling HSN development is at the time of HSN differentiation (M. Finney, Ph.D. thesis, MIT, 1987) and since unc-86 protein is expressed in the HSNs at that time ( Finney and Ruvkun, Cell 63:895, 1990). To understand how egl45 and sem4 act to control HSN differentiation, we are characterizing them in both genetic and molecular studies. Our genetic studies have concentrated on sem4. We have identified seven sem4 alleles which define three phenotypic classes: weak (nl 378), strong (nl 971 and four others) and lethal (n2088). n2088 fails to complement a number of linked lethal complementation groups and is therefore probably a deficiency; we are currently testing whether n2088 spans sem4. Strong sem4 alleles affect many other cells in addition to the HSNs, including those of the nervous system, musculature and hypcderm. Staining experiments using antibodies directed against the unc-86 protein have shown that the pattern of unc- 86 expression is altered in sem4 mutant animals (M. Finney, personal communication), suggesting that sem4 controls the expression of this putative transcription factor in some cells. For example, mutation of sem4 blocks the increase in unc-86 expression observed in the HSNs at the time of their differentiation. Since unc-86 is expressed only in neurons, however, the function of sem4 in non-neuronal cells presumably does not involve unc-86 but could involve other cell type- specific transcription factors. Our efforts to clone egl45 and sem4 have been greatly aided by the fact that both genes lie within mapped contigs. We have rescued both mutants in germline transformation experiments. egl45 is rescued by an 8 kb subclone of the cosmid T17D7 from the cluster on LG III. sem4 is rescued by the overlapping cosmids E02H2 and C09F2 from the cluster on LG I. Experiments in which both of these cosmids were injected together suggests that the complete sem4 gene includes DNA both to the left and to the right of the 18 kb region of overlap between the two cosmids. We are now attempting to identify transcripts and cDNAs corresponding to egl45 and sem4.

Aubry, G. et al. (2019) International Worm Meeting "An automated droplet-based platform for behavioral screens in C. elegans."

Aubry, G., Lu, H., Milisavjlevic, M.

[International Worm Meeting, 2019]

Screens in C. elegans can be a challenging task when analyzing behavioral response to chemical stimulation for large libraries or genetic screens. Major challenges reside in designing an efficient method to screen large number of chemicals, recovering animals on demand, and minimizing reagent consumption. Here we present a platform for screening C. elegans behavior in a high-throughput manner using droplet microfluidics. We have integrated on the same device several modules that enable precise manipulation of single nematodes. First, an encapsulation module allows for trapping animals in microdroplets, which provides a robust way to transport animals through the microfluidic network. Second, we have designed two microfluidic systems allowing for precise control of the animal's chemical environment. One system merges droplets, allowing for creating mixtures, while the other one exchanges the content of a droplet with another, allowing for full control over the worm's chemical environment. Finally, using on-chip valves, real-time image processing, and a custom LabVIEW program, the operation of the platform is fully automated. We have demonstrated the operation of the platform with a throughput of 100 worms per hour when monitoring behavioral response for 30 s and, as a proof-of-concept, we have studied male response to pheromones. In addition, active control over droplets allows for recovery of animals on demand. This platform can handle adult animals and can be adapted as well for more challenging situations, such as handling first developmental stage larvae. Finally, we demonstrate ultra-low reagent consumption with 100 nL of reagent per animal, which will facilitate screening libraries of expensive or rare compounds.

M Pilon et al. (1999) International C. elegans Meeting "C32E8.7, a C.elegans homolog of the diabetes autoantigen ICA69"

M Pilon, HM Dosch, AM Spence

[International C. elegans Meeting, 1999]

Type I diabetes is an autoimmune disease in which the immune system targets antigens in the insulin-producing pancreatic beta-cells. The Islet Cell Antigen of 69 Kd (ICA69) was discovered by screening islet cell expression libraries with serum from diabetic rats or human patients. The protein exhibits no significant homology with proteins of known function, except for the presence of a putative coiled coil motif possibly implicated in protein-protein interactions. Based on its amino acid sequence, ICA69 is predicted to be a cytosolic soluble protein. Only one ICA69 gene seems to exist in mouse (478 aa), rat (480 aa) and human (483 aa); these exhibit 87% overall identity. In mouse, the ICA69 protein is expressed in most organs, but is most abundant in tissues rich in neuroendocrine or secretory cells such as brain, pancreas and stomach mucosa. The C32E8.7 (418 aa) predicted gene encodes a C.elegans homolog of ICA69 (33% overall identity with human ICA69, including a stretch of 280 aa with 58% conservation). Using a GFP reporter in transgenic C.elegans , we found that the C32E8.7 promoter is specifically expressed in all C.elegans neurons; the timing of promoter activity coincides with the appearance of differentiated neurons during embryogenesis (~400 minutes). Because: 1) most diabetes autoantigens are components of the insulin secretory granules; 2) The high levels of ICA69 expression in neuroendocrine/secretory tissues; and 3) the C.elegans homolog of ICA69 is expressed in differentiated neurons, we hypothesized that ICA69 is involved in regulated exocytosis which mediates, for example, insulin secretion by pancreatic beta-cells and neurotransmitter release by neurons. In collaboration with Prof. Plasterk (Amsterdam, NL), we have isolated a C32E8.7 mutant (NL2003) in which nucleotides -395 to +2276 (262 codons) have been deleted. The NL2003 mutant exhibits no overt phenotype. However, we have observed a slight shortening of life span, and preliminary experiments suggest that the mutant is slightly resistant to aldicarb, an inhibitor of acetylcholinesterase. This later observation would support our hypothesis that ICA69, and its C.elegans homolog, is involved in exocytosis.