Jonathan Pettitt et al. (2001) International C. elegans Meeting "A family of beta-catenin interacting proteins involved in hypodermal morphogenesis"

Hendrik C Korswagen, Jonathan Pettitt

[International C. elegans Meeting, 2001]

In both vertebrates and Drosophila a single protein, beta-catenin/Armadillo, is a key component of both Wnt signalling and cadherin-mediated cell adhesion. In C. elegans the signalling and adhesion functions of beta-catenin appear to have been distributed between separate proteins. HMP-2 appears to function exclusively in cadherin adhesion complexes, while BAR-1 and WRM-1 act in Wnt signalling pathways. We have identified three genes whose products interact with both BAR-1 and HMP-2 in yeast two hybrid screens. These genes have been designated bin-1 , -2 and -3 ( b eta-catenin in teracting). The three genes encode related gene products that do not show any significant similarities to other non-worm proteins in the publicly available sequence databases. In order to determine the role of these genes we have examined the effect of depleting their function using RNA interference. bin-1(RNAi) resulted in variable defects in hypodermal cell positioning, particularly in the seam and ventral hypodermis, often resulting in ventral enclosure, or elongation defects. Neither bin-2(RNAi) nor bin-3(RNAi) resulted in an obvious mutant phenotype, and neither did the double RNAi of both bin-2 and bin-3 . However, double RNAi of both bin-1 and bin-2 resulted in an enhancement in the penetrance and severity of the defects observed with bin-1(RNAi) alone. RNAi of bin-1 and bin-3 resulted in a synthetic phenotype not seen in bin-1(RNAi) animals. These embryos all arrested with very pronounced defects in the pattern of hypodermal cell junctions, and never initiated ventral enclosure. These results demonstrate that bin-1 shares functions in regulating hypodermal patterning and morphogenesis with bin-2 and bin-3 . We do not know how these gene products act to regulate hypodermal development. They do not appear to regulate the activities of HMP-2 or BAR-1, since the mutant phenotypes of these genes do not cause the same phenotypes. We are currently attempting to determine whether the products of the bin genes can interact with other Armadillo repeat containing proteins, such as WRM-1, APR-1 and the C. elegans homologue of p120 catenin, as well as examining where they are expressed.

Chenggang Zhang et al. (2008) "Estimation of error rate of the genome-wide RNAi screening in Caenorhabditis elegans by analyzing non-specificity amplification of the PCR primers using the PSC program"

Wubin Qu, Zhiyong Shen, Chenggang Zhang, Bin Ning

[2008]

"Abstract: RNA interference (RNAi) is an efficiency and widely used technique in the functional genomics research. Genome-wide RNAi screening library for Caenorhabditis elegans is commercially available to advance large-scale analysis of the function for most (86%) of the 19,000 predicted genes. Although the RNAi screening library was constructed based on polymerase chain reaction (PCR) without directly sequencing to validate the nearly 16,000 gene sequences, however, the error introduced by PCR will probably occur during the preparation of the library and in turn will result in interfere of other unexpected genes. By using a recently developed computer program named Primer Specificity Checking (PSC, http://biocompute.bmi.ac.cn/PSC), the PCR primers for the 16,000 genes were analyzed carefully to determine whether they will result in mis-targeting the expected genes. Results indicated that at least 12.5% of the PCR primers have predominant non-specific amplifications. Most of the typical non-specific amplifications were caused by the primer-pairs capable to amplify different target genes in different chromosome location, while a minor proportion of the non-specific amplifications were caused by the self-amplification of only one primer. This will result in nearly 2,000 genes failed in the RNAi experiment. In conclusion, we suggested the user to check the primer sequences to avoid the error caused by PCR during the genome-wide RNAi screening in Caenorhabditis elegans. Keyword: Genome-wide RNAi screening; non-specificity amplification; Primer; PCR; PSC *Correspondence should be addressed to CZ (Email: zhangcg@bmi.ac.cn; Tel: +8610-66931590. Fax: +8610-68169574)."

Suzan J Holt et al. (2001) International C. elegans Meeting "A bin's-worth of bounty: SAGE data mining in dauer larvae and mixed-stage transcript profiles"

Suzan J Holt, Donald L Riddle, Steven J Jones

[International C. elegans Meeting, 2001]

What is the constitution of a survivor? Caenorhabditis elegans can endure adverse environmental conditions by entering the developmentally-arrested dauer stage. Metabolic changes in the dauer larva reflect the fasting survival period, which may extend to 12 times the normal life span. We compared gene transcript profiles between dauer larvae and non-dauer (mixed-stage) populations to further characterize the dauer state. Serial Analysis of Gene Expression (SAGE) enabled the identification of extant gene transcripts and a quantitative estimate of their respective abundance by correlations between predicted genes and short sequences (tags) from dauer and mixed-stage cDNA libraries. The number of SAGE tags per tag-transcript correlate indicates the abundance of the transcript in the dauer or mixed-stage profile. The existence of more than one unique tag per gene can be due to alternate transcripts. After exclusion of single occurrence tags, 9304 unique SAGE tags (7642 genes) constituted the dataset from which a dauer profile (7676 tag-transcript correlates, 1251 dauer specific) and mixed-stage profile (8053 correlates, 1628 mixed-stage specific) were derived. When tag-transcript correlates were grouped by transcript abundance classes (bins) in doubled increments, frequency distributions for the two transcript profiles were exponential. A negative log linear relationship between the transcript abundance bin and the number of transcripts in each bin was observed in both the dauer and mixed-stage profiles. A significantly greater variance in the dauer profile compared to the mixed-stage profile is explained, at least in part, by differing variances between pooled bins in the tails of the two distributions, where transcript abundance is high. Specific transcripts within bins can be ordered to show contrasts in the dauer versus mixed-stage profiles, by either abundance differences or ratios. The exponentially binned classes of transcript abundance not only provide manageable subdivisions for comparisons of dauer and mixed stages, but may reveal more global factors that govern the C. elegans transcriptome, such as gene location relative to condensed versus open chromatin regions.

Elizabeth Kimberly et al. (2003) International Worm Meeting "Molecular genetic characterization of a putative inhibitor of CEM cell death"

Ning Pan, Ding Xue, Ute Meisel, Erin Peden, Elizabeth Kimberly, David Kokel, Beryl Hatton

[International Worm Meeting, 2003]

Programmed cell death (PCD) generates sexual dimorphism by eliminating sex-specific cells or organs that are not needed in a specific sex of a species. We are interested in identifying genes that regulate sexually dimorphic PCD in C. elegans. Four male-specific cephalic companion neurons (CEMs) and two hermaphrodite-specific neurons (HSNs) undergo sex-specific PCDs in C. elegans. Although all are born embryonically in both males and hermaphrodites, the CEMs survive only in males and the HSNs survive only in hermaphrodites. The regulation of sex-specific PCDs in C. elegans offers an ideal paradigm for understanding the mechanisms that activate PCD in specific sets of cells. In C. elegans males, the CEMs are located between the two bulbs of the pharynx: two on the dorsal side and two on the ventral side. We have used a transgenic strain, pha-1(e2123ts); him-5(e1490); syEx313 (kindly provided by Maureen Barr and Paul Sternberg), in which GFP is specifically expressed only in CEMs and a few tail neurons in males, to screen for mutations that cause abnormal CEM PCD patterns. From a screen of 8500 haploid genomes, we isolated four mutations that affect CEM PCDs in males and nine mutations that inhibit CEM PCDs in hermaphrodites. We are most interested in the mutant rsd-6(sm130) (regulator of sex-specific death). In sm130 mutant males, both the dorsal CEMs and ventral CEMs undergo ced-3-dependent ectopic PCD. However, the dorsal CEMs are more strongly affected: only 8% of dorsal CEMs are present compared with 47% of ventral CEMs that survive. In sm130 hermaphrodites, CEMs undergo PCD normally. Since sm130 is a recessive mutation, it may affect a cell death inhibitor that regulates sex-specific CEM death. Genetic epistasis analysis suggests that rsd-6 acts downstream of tra-1, the terminal regulator of the sex determination pathway, and sel-10, a regulator of both CEM and HSN PCDs (see Ning Pan's abstract), to regulate sex-specific CEM deaths. Three-factor mappings and single nucleotide polymorphism (SNP) mappings placed rsd-6 in a two cosmid interval on the left arm of LG X. We found that one of the cosmids robustly rescues the sm130 phenotype. We have localized the rescuing activity to a region of the cosmid that encodes a homeobox protein. Further molecular and biochemical characterization of the rsd-6 gene should help reveal how sexually dimorphic apoptosis is regulated and executed in nematodes and may shed light on the regulation of sexually dimorphic cell deaths in general.

Tod R Thiele et al. (2003) International Worm Meeting "The homeobox gene ttx-3 is required for normal chemosensory step-responses in C. elegans."

Tod R Thiele, Shawn R Lockery, Adam C Miller

[International Worm Meeting, 2003]

The sensorimotor transformation underlying C. elegans chemotaxis has recently been defined by recording the behavior of unrestrained worms in a new step-response assay (Miller et al. IWM 2003). A sudden increase in NaCl concentration (an "upstep") increases the probability of forward swimming ("run" behavior), whereas a sudden decrease in concentration (a "downstep") results in the two-part fixed action pattern "turn-and-run". As a first step in investigating the neuronal mechanism of the chemotaxis sensorimotor transformation, we studied the effect on step-response behavior of disrupting the function of interneuron AIY. AIY receives direct synaptic input from the NaCl chemosensory neuron ASE. AIY function was disrupted using the ttx-3 mutation, which alters axonal outgrowth in AIY and copies the effect of AIY ablations in thermotaxis (Hobert et al. Neuron 19, 345-357). Behavior of individual worms was scored by noting the times of transitions between three main locomotory states (forward, reverse, omega turn). Responses were analyzed in terms of the probability of each state in 10 sec bins for several minutes before (baseline) and after the step (test). In each bin we also computed the rate constants for transitions between states. We made three main observations. (1) The ttx-3 mutation had little or no effect on baseline rate constants, implying that AIY does not significantly regulate locomotory state in the absence of chemosensory stimulation. (2) The ttx-3 mutation virtually eliminated the run that normally follows upsteps in wild type animals. Intriguingly, this effect was confined to upsteps, as the response to downsteps was essentially normal. This result suggests that interneurons in the chemotaxis network may comprise functionally distinct modules, for regulating upstep responses versus regulating downstep responses. (3) Inspection of the time courses of the rate constants during baseline and test periods in wild type animals showed a prominent spike in the reverse-to-forward rate that was absent in ttx-3 animals. There was comparatively little effect of the ttx-3 mutation on the time courses of the other rate constants. Thus the loss of the reverse-to-forward spike is the main reason for the absence of runs following upsteps in ttx-3 mutants. This result constrains models of how sensory input regulates locomotory switching in C. elegans. We are testing by laser ablation the hypothesis that AIY specifically regulates upstep responses. Supported by NIH (MH51383)

David M Raizen et al. (2003) International Worm Meeting "Long-term observations of L1 rest/activity cycles"

Joan C Hendricks, Allan I Pack, David M Raizen, Meera Sundaram

[International Worm Meeting, 2003]

Diurnal environmental cues consisting of light and temperature changes play an important role in control of animal behavior. Behavior of food-deprived L1s, a long-lasting developmental state, may be particularly adaptive to daily changes in conditions, and may therefore be a useful system to study diurnal changes in behavior. Using time-lapse video analysis of L1s in the absence of food, we have been observing movements of L1 for several days. We transfer ~3000 worms in solution onto an agar surface of a 3 cm plate. We videotape the worms at 30-minute intervals for 2 minutes under red light, and then analyze the videos in time-lapse mode. For some experiments, the worms are exposed to white light for 12 hours alternating with darkness for 12 hours. We have observed two distinct behavioral states. In one state, the worms are swimming rapidly across the agar surface with few changes in direction. This state is reminiscent of the roam state defined for adult worms in the presence of food by Fujiwara et al (Neuron 02). In the other state, which is reminiscent of the dwell state defined by Fujiwara et al, the worms make little to no net movement over the two minutes of observation. We define worms that had made at least one net body length movement in the two-minute observation bin as active and other worms as resting. L1s populations remain active continuously for the first day after transferring from solution onto an agar surface. After approximately one day of continuous activity, there is decay in worm activity over the next several days. This decay in activity is unlikely to be explained simply by fatigue or sickness of the worms for the following reasons: (1) the rest state is not permanent. (2) Survival of L1s maintained on an agar surface in the absence of food does not drop significantly until 7-10 days after plating, yet activity drops significantly by the second day after plating. (3) After spending a week on one agar plate, the fraction of active worms drops to approximately 0.3. If these worms are then washed off this plate and transferred to a fresh plate, the fraction active increases immediately back to nearly 1.0. We suggest that this activity decay process reflects habituation to a novel environment. Our second observation of activity relates to the rest/activity patterns within each day. We have found the when the day is divided into 12 hours of light and 12 hours of darkness, worm activity is significantly higher during the light (L) phase than during the dark (D) phase. FFT analysis of rest/activity cycles reveals a peak at a period of 24.5 hours, indicating that there is a diurnal rhythm of rest/activity under L:D conditions.