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We are isolating and distributing deletion mutants to the community using a TMP-UV method (Gengyo-Ando and Mitani, 2000). To collect high-quality mutants, we used to examine the presence of wild-type DNA sequences when deletion sizes are longer than 1 kb. However, after a time-point, we changed the criteria shorter than before: 500 bp. When deletion sizes are shorter, we can easily identify such a rearrangement as we do "homo-hetero" tests (as seen by WT band in lane 1 in addition to internal band in lane 2 of type #2 mutant in Figure 1). Most of the mutants caused by rearrangements can be excluded this way and not posted publicly. We guess, however, there may be still a small number of such rearrangement artifacts in our mutant collections in earlier tm alleles (before tm2000). We re-examine the rearrangements when necessary and continuously improve the quality of the collection; we do the re-examination when we find strains that turned out to be homozygous viable, which were regarded as homozyogous inviable and did not examine the internal primer test before. We withdraw alleles when we find problems and try to replace them by isolating new alleles. We hope if people find such cases, please let us know.

Chemotaxis bioassays are necessary for identifying and characterizing signals that cause attraction or repulsion responses in C. elegans (Ward, 1973). One common method for performing bioassays is to paralyze worms with sodium azide once they reach the material and count the numbers in each area. Another is to record the entire bioassay, which allows you to extract additional data such as detailed spatial information (useful in lawn leaving assays), worm speed, and reversal frequency (Srinivasan et al., 2008; Buckingham and Sattelle, 2008; Bendesky et al., 2011). The downside to this method is its low throughput that is limited by the number of microscopes. We present here a multiple camera recording system using six webcams mounted above a custom made light box which allows the recording of six bioassays simultaneously (Fig. 1). These recordings are processed using a custom MATLAB script which outputs a histogram showing the positions of all worms over the course of the assay. Multiple histograms are summed together using image registration. Test assays were performed using diacetyl (a known attractant), verifying its usefulness in attraction bioassays (Fig. 2).

This apparatus is still under development, and we welcome suggestions from the community on how to improve it. Specifically, we have been unable to connect six cameras to one computer due to limitations in USB bandwidth and are using 2 computers instead. We would also like to be able to record assays with L4 and younger worms, which is currently difficult due to the low contrast of the videos. General methods are described below; details will be described in a further publication.

WormBox Construction

Six Microsoft LifeCam Cinema 720p HD webcams are mounted in a custom holder made of nylon above a PVC lightbox outfitted for brightfield microscopy. The reflector is white photo-quality paper, positioned at an approximately 45° angle. The light was constructed using 7 Philips Luxeon K2 Cool White LEDs. Four cameras are connected via USB 2.0 to a Windows 7 x64 computer with 16GB RAM and two cameras are connected to a Windows XP computer with 4GB RAM. The images from each camera are captured with the MATLAB Image Acquisition Toolbox and saved as often as possible (around 1 Hz).

Video Analysis

The background, which is calculated using principal component analysis, is subtracted from each image. The subtracted images are binarized and cleaned to remove stray pixels and connected components which are too small to be worms. The resultant series of binarized images are summed to generate a histogram, or a record of the locations of all worms across time. Multiple assays are aligned using the scoring regions as control points, and summed. All processing is done in MATLAB.

Acknowledgments

We thank Ryan Newsome and the Florida State University Machine Shop for help designing and for constructing the light box and the UF Center for Smell and Taste for machine shop funds (NIH P30 DC010364). We thank Chaevien Clendinen and Circe Lassegue for help with the project. This work was supported by 1R01GM085285 and 3R01GM085285-01A1S1.

C. elegans are a historically relevant model for many biological and chemical laboratories. Microscopy with C. elegans has been explored previously, and our lab focuses on the use of fluorescent microscopy with live nematodes. Imaging live C. elegans raises several issues such as how to treat the samples, how to immobilize the subject, and how to quantify the fluorescence. Previous studies imaging C. elegans mostly focus on fixing the worms with chemicals and do not allow for live imaging. Other studies have used a slide and coverslip method for immobilization with minimal sample volume or the addition of agarose pads to the slide. In our experience, the C. elegans move too fast for fluorescent imaging or their bodies are crushed under minimal volume conditions. Ethanol is a known locomotion inhibitor for C. elegans at concentrations near 400mM (McIntire, 2010) and it seemed to be a likely candidate for immobilizing live nematodes. Another advantage of this technique is that it allows for the recovery of live nematodes.

C. elegans are grown as plate culture at 19 ^oC for 6-7 days, and then synchronized for a uniform developmental stage harvest as described in Wormbook (Stiernagle, 2006). Plates are treated with fluorescent dyes in M9 buffer in the presence of E. coli for 20 minutes. C. elegans are removed from the plates and 400 mM ethanol is added to the solution for 5 minutes to anesthetize nematodes. A five minute treatment with 400 mM ethanol immobilizes C. elegans for up to twenty minutes of imaging. From each sample, 10 µL of the solution is pipeted onto a Thermo Scientific ColorFrost Plus Slide™ and covered with a Columbia Calibre® glass coverslip. The C. elegans are imaged with an Olympus inverted microscope IX81/DP71, using appropriate fluorescence filters. To obtain quantitative microscopy results, images are analyzed for pixel intensity using ImageJ Software provided by NIH.

We have developed a protocol that greatly speeds preparation of C. elegans samples for analysis by immunoblotting. A key point of simplification in the protocol is standardizing the age and numbers of nematodes used for the studies. Our immunoblot analyses use 15 to 50 age-synchronized nematodes per well of 10 or 15 lane mini protein gels (Invitrogen). The exact number varies depending on the nematode age, the antigen abundance and the quality of the antibody. However, to provide investigators a benchmark from which they can further optimize their studies, we provide three examples using Actin as the antigen and hermaphrodites as the source material: L4/35 nematodes, adult day1/30 nematodes, adult day3/20 nematodes. Use of a defined number of age synchronized nematodes enables us to load exactly the same amounts of total protein from multiple samples with very minimum variation between the gel wells.

The sample preparation does not require sonication or any other mechanical method to crush the nematodes. Synchronized adult animals first are cleaned by transferring to a NGM plate with no bacteria. Next, 15 to 50 nematodes are picked and placed into 20 μL of DDH₂O that is contained in the cup-like part of a severed Eppendorf tube lid (Fig.1). Once the nematodes are released in the water droplet, the lid is put on an intact Eppendorf tube containing 20 μL of 2X sample buffer already pipetted at the bottom of the tube (Fig. 1). The nematodes are spun into the 2X sample buffer (see below) for one minute at 12,000 RPM. The pelleted sample with nematodes is then flash frozen in an ethanol/dry ice bath; at this point the frozen sample can be stored in a -80⁰C freezer until one is ready to run the gel. The volumes used in the protocol can be adjusted to match the sample size (for example, 10μL H₂O+10 μL 2X sample buffer for 15 adult day1 nematodes).

Prior to running the samples in a mini gel, the samples are incubated at 95⁰C in a heating block for five minutes, followed by five minutes of cooling at room temperature. The samples are then heated again for the second time at 95⁰C for five minutes and can be loaded onto a gel. The subsequent immunoblot protocol varies depending on the particular antigen and antibody used. In our laboratory, proteins in the gels are transferred to a PVDF (BioRad Inc.) filter using NuPage transfer buffer with 10% Methanol and 0.01% SDS at 20 volts overnight or a shorter period (3 to 5 hrs) until no marker protein is left in the gel. Incubation with the primary antibody is commonly performed in PBS containing 4% blotting grade milk with 0.025% Tween-20 for two to twenty four hours in a cold room (4⁰C), depending on the antigen affinity of the antibody. Incubation with the secondary antibodies is done for two hours at room temperature with three subsequent washes (5 min X 3) with PBS containing Tween-20 prior to treating with ECL solution.

2X Sample buffer: 100mM Na-Tricine pH 7.8, 100mM DTT, 14% W/V Glycerol, 4% LDS, 0.05% CHAPS and 0.002% Bromphenol Blue (can be added later if protein concentration is to be determined for the lysates). The sample buffer can be frozen in aliquots in -20⁰C for long-term storage.

2μL HALT (Protease inhibitors cocktail, Sigma) should be added to 98μL 2X Sample buffer prior to mixing with nematodes.

Allele-specific PCR (AS-PCR) is an inexpensive and fast method to discriminate genotypes based on single nucleotide differences. It relies on the requirement of Taq polymerase for valid base-pairing between the template and the 3’-most nucleotide of allele-specific primers (ASPs). In its simplest form, an AS-PCR combines three primers in one reaction: two ASPs, each of them matching one of the two allelic nucleotides at its 3’-most position, and a common reverse primer. For the single-tube reaction, at least one of the ASPs is tagged to distinguish the alternative products, e.g., a size tag of 5’ non-specific nucleotides allows for separation by gel electrophoresis. Unfortunately, the method requires extensive optimization of conditions to obtain sufficient specificity for unambiguous genotyping, which has hindered its wide-spread use in genotyping SNP alleles and is preventing its use in SNP mapping.

We realized that the amplification process limits specificity of single-tube AS-PCR because relatively rare events of mispriming generate allele-switched amplicons that are propagated during subsequent cycles (Fig. 1). We found that incorporating additional mismatches into the ASPs that make them different from both the template and one another at the third-last position prevents this problem because a single round of amplification now results in amplicons with a perfect match to one ASP, but two mismatches to the other (which is not achieved by introducing a common mismatch into the ASPs as sometimes suggested). Thus, mispriming is effectively limited to the original template and the inherent specificity of the polymerase for the matching primer is preserved over any number of cycles. We routinely use this improved snPCR (for PCR with single nucleotide specificity) in the Alcedo lab to follow genotypes during crosses and found it to be just as robust and reliable as standard PCR (two examples are given in Fig. 2). snPCR works well with standard worm lysates and optimization of conditions is usually not required. For separation of the alternative products we use size tags of 25-30 non-specific nucleotides on one of the two ASPs and 3% agarose (Nusieve 3:1 for the separation of nucleic acids < 1kb) gels. To achieve good separation we design the primers to amplify fragments of < 500 bp. We have also successfully used Cy5/Cy3-labeled ASP pairs to distinguish products based on fluorescence rather than size and read out genotypes from standard gels with a fluorescence scanner. Avoiding interference between the size tags and overcoming the product size limitation this approach should enable the multiplexing of snPCRs and should make the method useful also for SNP mapping. Since relative allele frequencies can be determined with snPCR it should be compatible with the bulked segregant analysis described by the Plasterk group (Wicks et al., 2001) and represent a convenient and fast alternative to allele-specific restriction digests.

During the preparation of this article, we found that the principle behind our snPCR has recently been described elsewhere (Gaudet et al., 2009). The authors state that any mismatches within the last 5 nucleotides of the ASPs - even if they differ in position between the two ASPs - can be chosen, provided they result in two versus zero mismatches after the first round of amplification. This finding is in good agreement with our mechanistic explanation and, if true, makes the design of the ASPs quite flexible.

Conversations at the 2011 IWM prompted me to share the following. A recent attempt at microinjection resulted in only a few F₁ transgenics (< 0.2 F₁s per injected animal) and no transmitting lines. The major cause was traced to the use of RNase in a newly-opened Qiaprep Spin Miniprep Kit. I have been routinely omitting addition of RNase to the P1 suspension buffer in the kit due to a previous failure to recover F₁s if RNase was used with modified alkaline lysis and ethanol precipitation (Birnboim and Doly, 1979), even with organic extraction. Using new plasmid DNA prepared with P1 lacking RNase, many F₁s and transmitting lines were recovered, though the efficiency, ~7 F₁s per injected animal, was still significantly lower than the ~25-29 F₁s per injected animal reported by Mello et al. (1991) at similar DNA concentrations (180 ng/μL; see Table 1). The optional PB rinse step in the Qiagen kit did not seem to improve the efficiency. Instead, it was found that Ethanol Precipitation (EP) of the Qiagen-prepared DNA increased recovery of transformed F₁s to an efficiency close to that of Mello et al. (1991) (Table 1).

The precipitation step significantly improved recovery of F₁s and transmitting lines with routine injections and, especially, recovery of insertions with the Mos-based Single Copy Insertion method (MosSCI; Frökjær-Jensen et al., 2008). The very similar but less expensive Fermentas GeneJET Plasmid Miniprep kit (#K0503; omit RNase, add EP) was also found to work just as well. It is acknowledged that others in the community may not be experiencing any issues with their miniprep DNA and that their experience might be completely different from what is reported here, for a variety of reasons. However, if some are experiencing low worm transformation efficiencies with spin column-purified DNA, the omission of RNase and addition of Ethanol Precipitation might be worth trying.

Table 1: Recovery of F₁s following microinjection.

(1) EP of DNA made with P1+RNase was not tried, as previously it was found that DNA made following alkaline lysis, RNase treatment, organic extraction and EP did not result in DNA preps that gave good worm transformations.

(2) EG4322 [ttTi5605 II; unc-119(ed3) III] animals were used for both sets of experiments. Injected animals were pooled together in groups, so it is not known if all injected animals gave transformed progeny.

(3) Scored by rescue of the unc-119 locomotory defect. The proportion of F₁s making transmitting lines was not scored.

(4) Plasmids pDP#MM016B [unc-119(+)] and pMM824 [unc-119::mCherry] were transformed into E. coli XL2-Blue. 4-mL overnights were grown in LB+carbenicillin (10 μg/mL). DNA was prepared using the Qiaprep Spin Miniprep Kit without RNase in the P1 buffer, and eluted in 50uL of EB (10 mM Tris pH 8.3). Agarose gels of DNA prepared without RNase suggest very little RNA (if any) survives the final elution step. DNA concentration was measured with a Nanodrop spectrophotometer and the injection mix made in EB to a final concentration of 90 ng/μL per plasmid in 10 μL.

(5) Ethanol Precipitation was performed by adjusting the volume of the original plasmid preparations to 200 μL with ddH₂O, adding 20 μL of 3M sodium acetate (pH 4.8) and 550 μL of 95% ethanol, placing at -20°C for 15’, pelleting at high speed for 10’, rinsing the pellet in 80% ethanol, and suspending in EB. Concentrations were adjusted to 500 ng/μL and a second injection mix was made in EB at the same DNA concentrations.

After years of dropping my worm pick on the floor, only to have it shatter because it was forged from a Pasteur pipette, I have started to use a metal-handled pick.  The problem with my previous attempts to use metal handles failed because it was difficult to cinch them onto the platinum wire. However, using thin stainless steel sheet metal as a shim, it is straightforward to adapt X-acto knife holders (Fig. 1A) as worm picks.

Obtain thin metal sheeting [e.g., stainless steel foil .0025-.005cm thick] (Fig. 1B) and cut into strips with scissors (Fig. 1C).

Fold metal into a WW shape to make eight layers (Fig. 1D) that can sandwich the wire in the middle.

Sandwich the wire with the sheet metal (Fig. 1F).

Insert sandwich into slot in handle (Fig. 1F).

Tighten the nut (Fig. 1G).

Flame and use.

Some of the handles come with screw-on caps (Fig. 1H), which protect the pick if you put it in a lab coat or pocket. Fancier handles are available (Fig. 1I-J).  The handles can be engraved for special gift editions.

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At WormBase, we are often asked if it is possible to install and run a local version of the website. Although certainly possible and very well documented, it's not recommended for three reasons. 1) The size of the databases require substantial download time that must be repeated on a monthly basis to maintain an up-to-date resource; 2) The site is complex and requires significant time to install and configure; moreover, it is constantly evolving. You'll need to commit time to keeping your site up-to-date; 3) Finally, the site requires a substantive compute environment along with concomitant system administration acumen required to keep everything up and running smoothly. If you still aren't dissuaded, please see the installation notes on the WormBase Wiki.

But now -- through the magic of cloud computing -- you can have your own WormBase up-and-running in a few minutes.

Required Steps

1. Establish an account on Amazon Web Services
2. Find and launch the WormBase Amazon Machine Image (AMI) of the version of your choice.
3. Connect to the newly launched server instance using your web browser.
4. Stop the instance when done to avoid incurring further charges.
5. Repeat steps 2-4 when a new version of WormBase is released.

Intended Audience

• Individual researchers or labs
• Entire departments
• Private research entities

Necessary Skills

• For launching an instance: none beyond using a web browser
• For more complicated data mining: command line expertise

Suggested Uses

• Access your own WormBase: speedy and it's private
• Data mining: all databases preconfigured; includes common tools like BioPerl
• Development: build new features using the WormBase web platform

Caveats

• This is NOT a free service. Read the pricing details carefully.
• Although we will release new AMIs for each release, your instances will not receive bug updates.

For more information and a detailed walkthrough of the process, please see An introduction to cloud computing for biologists.

In common with many labs, in the past we routinely used DAVID to look for over-representation of functional classes in lists of genes. Unfortunately, the data in DAVID hasn't been up-dated since September 2009. As an alternative, there's a stand-alone version of DAVID called EASE. This allow labs to use their own lists. A post to the forum last year asking whether anyone maintained up-to-date EASE lists went unanswered, so we decided to make our own. That's when we realized that there are three challenges. One is simply having a user-friendly interface to enter any new gene list, with the appropriate citation information. The second is converting any gene list culled from the literature into a coherent form. This is made more difficult by the fact that one still finds gene lists in published papers that (i) contain an apparently random mix of different types of gene identifiers and (ii) don't say which WormBase WS version was used. The third is that with changes in predicted gene structure (genes being split, killed, fused, etc), lists become increasingly inaccurate with time.

We therefore made a first tool, the WormBase Converter. It can automatically detect different IDs (Transcript Name, Gene Name, WormBase ID, etc.) and convert them into the type of ID you need. It also allows the conversion between WormBase releases. You can convert a Gene ID from a specific release (e.g. WS170) into another release (e.g. WS220 for a conversion, or WS160 for a reverse-conversion).

As WormBase Converter was originally made for use with EASE, we didn't design it to give a correspondence table, but are currently working on adding this as an option, so that there will be the possibility of having an output table listing in one column the input genes and in an adjacent column the list of genes in the required output format. There are stand-alone WormBase Converter versions for all common platforms (Unix, MacOS, Windows) available on Sourceforge.org. As keeping it up to date does take some effort, due to the need to correct the occasional Wormbase annotation anomaly, we also offer a client version that sends requests to the WormBase Converter server that we maintain at the CIML. We'd be delighted if anyone else would like to host this service!

We also made EASE Manager. This facilitates the entry and management of gene lists into EASE, but more importantly, interfaces with the WormBase Converter so that the genes that make up any list are kept up to date. Installation instructions and full documentation can be obtained from our website. We welcome feedback on these tools, which we also described in a paper earlier this year (Engelmann et al., 2011).

The modENCODE 'Integrative Analysis' paper (Gerstein et al., 2010) described 304 unusual transcription factor binding site regions which they named 'Highly Occupied Target (HOT) regions'. These short (~400 bp) regions bind to 15 or more transcription factors and are not enriched in transcription factor motifs when compared to the normal specific binding sites. The HOT regions are associated with genes which are highly expressed and which are more likely to be essential compared to other genes. The HOT regions are included in WormBase as the features 'WBsf216780' to 'WBsf217083', inclusive.

The HOT regions tend to be associated with the 5' region of operons. This association was not commented upon by the authors of the modENCODE paper (Gerstein et al., 2010).

82 of the HOT regions are located within 2 Kb of the 5' end of operons. 20 of these are within 2 Kb of two operons flanking them on both strands. Operons sometimes have genes at either end which have not been included in them for lack of evidence, or have genes added to them which should not be included. If the distance from the start of the operon is extended to 10 Kb to allow for the uncertainty in the start position of an operon, then the number of HOT regions located near the 5' end of an operon increases to 143, and the number of these with two flanking operons increases to 40.

Assuming the null hypothesis that HOT regions should associate with the genes at the 5' end of operons as frequently as with any other coding gene, the binomial distribution gives a significant result for the observed association (p-value < 2.2e-16).

HOT regions may be useful for locating operons which have not been curated so far. Several such potential operons have been seen during a cursory inspection of those HOT regions which are not near a known operon. HOT regions will not however be used by the WormBase curators as evidence for the existence of a nearby operon because HOT regions are just as likely to control genes with a property like being constitutively expressed (which could preferentially include operon genes) as they are to be specific markers for the 5' end of operons.

Automated tracking of fluorescently labeled nuclei in 4D data sets has been used for several years for cell lineage tracing (Bao et al., 2006) and mapping gene expression patterns in C. elegans (Murray et al., 2006). Fully automated tracking of nuclei during late morphogenetic movements (i.e. above 350 cells) has remained challenging owing to the increasing density of nuclear packing in the later embryo.

We have developed a new tool for tracking labeled nuclei that avoids the increasing error rates associated with fully automated lineaging. Our approach is semi-automated: starting with a curated map of all nuclei in a 4D frame at any given time point, our software conducts local searches for nuclei at the next time point. Any nucleus that appears to have moved less than a threshold distance (0.75 of its radius) is assumed to be the same nucleus as in that position at time t. Nuclei that have moved greater than the threshold distance, or new nuclei arising by division, are flagged to be curated manually.

Using this approach we can trace all nuclei in individual embryos up to the 1.5-fold stage (670 nuclei; Figure 1), after which muscle movements make tracking difficult. It takes approximately 8 hours to track nuclei up to the 350-cell stage, and 80-100 hours to the 1.5-fold stage. Our software generates visualizations of the 4D data sets that can plot nuclear velocities and trajectories over chosen intervals. Our software has been used on 4D movies of histone-GFP (zuIs178) labeled nuclei acquired on a Zeiss LSM510 confocal and saved in the Zeiss .lsm format. We have been able to track nuclei in other confocal 4D data sets, the limiting factor being blurring of nuclear GFP signal at the top of a z-stack as nuclear density increases. In principle our approach can be applied to any sample in which many objects are being tracked over time. Our code is available at http://sourceforge.net/projects/nucleitracker4d/. NucleiTracker4D v2.0 requires a computer with at least 4 Gb of RAM as well as Matlab and its image processing toolbox. We welcome feedback from the community.

In reverse genetics RNAi that is suitable for gene-disruption is an outstanding method for knockdown of gene function. In Caenorhabditis elegans feeding RNAi is the most convenient. However, when knockdown of two genes (double knockdown) is carried out injection RNAi or soaking RNAi is usually adopted with a mixture of dsRNAs produced in vitro. That is due to less reliability of traditional double feeding directed to two genes as follows: in some cases, only one gene’s function may be significantly inhibited, or both genes may be only slightly knocked down by feeding with a mixture of two sorts of RNAi bacteria at the same time.

Therefore, we attempted to establish an efficient method of feeding RNAi for multiple-knockdown. We produced bacteria yielding distinct dsRNAs bound to each other. We selected oma-1 and oma-2 genes that are functionally redundant to each other. Each cDNA was amplified, sub-cloned into a T-vector (pGEM-T EASY, Promega), and then separated from the vector by using EcoRI. The resulting fragments were introduced into the RNAi vector L4440, yielding three sorts of RNAi plasmids: oma-1, oma-2, and both of them. We carried out double feeding for oma-1 knockdown and oma-2 knockdown, and single feeding for oma-1/oma-2 knockdown. Quantitative RT-PCR and observation of phenotypes revealed that our method for double knockdown by single feeding was much more effective than the traditional double feeding. This result has been describe in our report (Gouda et al., 2010).

Next, we applied our method to triple knockdown. We chose gfp gene in addition to oma-1 and oma-2 genes. We constructed an RNAi plasmid including cDNAs for these three genes as described above and carried out single feeding on C. elegans TJ356 strain, which expresses GFP constitutively. Observation of phenotype and GFP fluorescence revealed that knockdown of these three genes was quite effective. Min and coworkers have also reported the same result (Min et al., 2010).

In addition, we asked if our method might be applicable to genes which function in nerve cells. We selected rrf-3 gene in addition to gfp gene and carried out single feeding on the TJ356 strain. GFP fluorescence in nerve cells could be seen, while almost all fluorescence of GFP in somatic cells disappeared. Continuation of the feeding on descendants could not decease GFP fluorescence in nerve cells. Anyhow, our convenient method for multiple-knockdown is quite useful for investigating gene’s function in C. elegans.

The copy number and the repetitive nature of transgenes are important factors that need to be considered when trying to recapitulate the expression of a given gene as close to its endogenous counterpart as possible. It has long been known that low complexity transgenic arrays, or arrays with very high copy number can be silenced, especially in the germline (Kelly et al., 1997). In an attempt to generate fluorescent reporters that faithfully reflect the expression pattern of the gene of interest, we and others have previously reported the use of fosmid-based transgenes (Tursun et al., 2009). Here, we analyze the composition of high complexity arrays, some of which show robust germline expression, providing guidelines that should prove useful to other C. elegans researchers.

We created transgenic arrays by injection of the specified DNA (fosmid, plasmid or PCR product of the locus of interest) at the indicated concentrations (Figure 1). In all arrays, except ntIs1 and otIs314, all component DNAs were linear. The integrated strains were generated by γ-irradiation and they were outcrossed at least twice. The strains carrying the extrachromosomal fosmid arrays were co-injected with the pBX plasmid containing the wild type copy of the pha-1 gene in a pha-1(e2123) mutant background strain (Granato et al., 1994). Genomic DNA was prepared from the transgene containing strains and analyzed by array Comparative Genomic Hybridization (aCGH) (Maydan et al., 2007). The arrays used contain 50-mer probes tiling the 100-Mb genome of C. elegans. A segmentation algorithm was able to identify all transgene components. The log₂ of the ratio between fluorescent intensities (array containing strain/wild type) was averaged over the area of the genome that was detected as being amplified and is shown as the “Mean log₂ ratio”. From this we estimated the number of copies of each component of the transgene, shown as number of copies per chromosome for the integrated transgenes, or number of copies per array for the extrachromosomal arrays (Figure 1).

Strains containing the ntIs1 transgene have been previously whole genome sequenced in our lab (Sarin et al., 2010). We used these data to calculate the copy number of the components of ntIs1 transgene by dividing the average sequencing depth of the transgene region with the average sequencing depth across all non-gap regions, and found 51 copies for gcy-5^prom::gfp (vs. 31 by CGH) and 13 copies for lin-15 (vs. 11 by CGH). Comparison of these numbers with those from the aCGH analysis supports the fact that in general estimation of copy number by aCGH is more accurate for log₂ ratios lower than +4 and there is probably an under-estimation of copy number for log₂ ratios higher than +4 since they fall in the non-linear range, near saturation in that case.

For the extrachromosomal arrays of the fosmid reporters, an injection concentration of 15-50 ng/μl resulted in an average of 8 fosmid copies per array. While the data show that there is not a perfect correlation between injection concentration and copy number one could try to reduce the injection concentration if lower copy numbers were desired. In our experience, even transgenes that are integrated at 11 copies per chromosome (22 in a homozygote animal) are still able to provide germline expression, as seen for otIs284 (Tursun et al., 2011).

In an attempt to identify temperature sensitive alleles that prevent transcription at the restrictive temperature, we have identified two ts alleles of the TFIID initiation factor subunit, taf-6.2. These alleles were isolated by M. Wallenfang in the Seydoux lab in a screen for temperature sensitive embryonic lethals (Golden et al., 2000). The strains JH873 (ax701) and JH686 (ax514) were thought to have ts defective transcription based on the fact that they 1) did not express an early zygotic reporter, and 2) when adults were shifted to 25°C the embryos arrested at a point similar to ama-1(RNAi) (Wallenfang et al., 2002; Powell-Coffman et al., 1996).

We first mapped the ax701 mutation in JH873 to chromosome IV, and positioned it between dpy-13 and unc-17 (8/9 Dpy non-Unc; 9/53 Unc non-Dpy). A candidate gene in this region that matched the phenotypes observed for ax701 was taf-6.2 (Y37E11AL.8). Sequencing of taf-6.2 in JH873 (ax701) identified 2 missense mutations (R144H, G459A) in the TAF6 domain (AA105-475) of the taf-6.2 coding region. During the course of mapping and sequencing ax701 it was found that ax514 did not complement ax701, suggesting that ax514 was also located in taf-6.2. Sequencing of taf-6.2 in JH686 identified a missense mutation (G421E) which was also in the TAF6 domain of taf-6.2. These mutations were not found in either an N2 strain or the parent strain mutagenized in the original screen, JH150. In addition, no mutations were found in non-coding regions of the taf-6.2 ORF of either strain. The strains were outcrossed six times each, and the lesions we identified following the ts embryonic lethal (Emb) phenotype in the outcross.

Finally, the Emb phenotypes can be rescued with a WT copy of taf-6.2. A construct containing the taf-6.2 ORF plus 200 bp upstream and downstream of the ORF (pBAB1) was injected into the outcrossed ax701 strain with PD118.20 (myo-3:GFP). Nine independent GFP⁺ rescued lines that could grow at the restrictive temperature (25°C) were obtained. The taf-6.2 transgene also rescued JH686 (ax514) to viability at 25°C.

In addition to the phenotypes mentioned above, further lines of evidence suggest that ax701 prevents transcription at the restrictive temperature. Embryos raised at the restrictive temperature failed to gastrulate, as is observed in ama-1(RNAi) (Powell-Coffman et al., 1996). In addition, shifted ax701 unc-17 embryos showed a lack of a transcriptional elongation marker, phosphorylation of Ser2 on the CTD repeat peptide (H5 antibody).

In summary, we have identified two temperature sensitive alleles of the transcription initiation factor, taf-6.2. While we have confirmed that mutations in taf-6.2 are responsible for embryonic arrest phenotypes that suggest that RNA Polymerase II transcription is shut down or severely compromised in these strains, we have not yet verified the transcription defect using high resolution or biochemical assays. Overall, we think these alleles will be important to the worm community as a way to modulate transcription in a time- and temperature-sensitive manner. We plan to make the unc-17 taf-6.2 outcrossed strains available to the community through the CGC [KW1973: taf-6.2(ax701) unc-17(e113) IV and KW1975: taf-6.2(ax514) unc-17(e113) IV].

To study the interaction of a small molecule with worm ribosomes, we modified a protocol for isolating yeast 80S ribosomes (Algire et al., 2002). N2 worms were cultured in S Medium with E. coli HB101 as described in WormBook (Stiernagle, 2006). Worms were separated from bacteria by sucrose flotation (Portman, 2006). The worms from one liter culture resulted in about 5 mL of pellet. 5 mL of ribosome buffer plus heparin and protease inhibitors (100 mM KOAc, 20 mM HEPES-KOH, pH 7.6, 2.5 mM Mg(OAc)₂, 1 mg/mL heparin, 2 mM DTT, 1X protease inhibitor cocktail) were added to resuspend the worm pellet, followed by mechanical homogenization with 0.7mm zirconia beads (BioSpec Products. Inc.). The lysate was centrifuged at 17,000 × g for 30 minutes at 4ºC. The supernatant was transferred to polyallomer Microfuge^® tubes and centrifuged at 400,000 × g for 20 minutes at 4ºC. The pellet was resuspened in 1.5 mL of high salt buffer plus heparin and protease inhibitors (100 mM KOAc, 20 mM HEPES-KOH, pH 7.6, 2.5 mM Mg(OAc)₂, 1 mg/mL heparin, 500 mM KCl, 2 mM DTT, 1X protease inhibitor cocktail), and the mixture was rocked for 45 minutes. After this high salt wash, the mixture was centrifuged at 20,800 × g for 10 minutes at 4ºC. The supernatant was loaded on the top of a 250 ml of sucrose cushion (100 mM KOAc, 20 mM HEPES-KOH, pH 7.6, 2.5 mM Mg(OAc)₂, 500 mM KCl, 1 M sucrose, 2 mM DTT). The mixture was centrifuged again at 400,000 × g for 20 minutes at 4ºC. The ribosome pellet was washed quickly with 500 mL of ribosome storage buffer (100 mM KOAc, 20 mM HEPES-KOH, pH 7.6, 2.5 mM Mg(OAc)₂, 1 mg/mL heparin, 2 mM DTT). The pellet was resuspened in 100 ml of ribosome storage buffer plus 2 ml of RNAseOUT^®. The mixture was flash frozen in liquid nitrogen and stored at -80ºC. The concentration of 80S ribosome were determined by the absorbance at 260 nm and using extinction coefficients of 5×10⁷ cm^-1 M^-1 (Algire et al., 2002).

This method provides active worm 80S ribosomes capable of being used in biochemical studies, such as binding experiment with small molecules. We were able to use this method to study the binding of synthetic natural toxins to worm ribosomes isolated from different strains. This method might also be applied to other biochemical studies of worm ribosomes.

We are interested in the C. elegans nociceptive response, and particularly in the involvement of neuropeptides, including opioids, in this response. C. elegans has five TRPV-type channels, osm-9 and ocr-1–4 (Xiao and Xu, 2011). Interestingly, we have previously noted that wild-type worms do not normally avoid capsaicin (consistent with previous findings, Tobin et al., 2002), yet worms DO avoid crude hot pepper extracts, the primary active ingredient of which is capsaicin. We also previously described that the worms avoid crude extracts of garlic, black pepper, hot mustard, and cloves, the putative active ingredients of which are allicin, piperine, allyl isothiocyanate, and eugenol, respectively (Sadaiappen et al., 2009). Of these, it had previously been determined that worms are neither attracted to or repelled by eugenol (Bargmann et al., 1993). We wished to test whether the other active components of our extracts were nociceptive agents for worms. We were particularly interested in the worms’ response to piperine, as this compound is known to act on the same TRPV1 receptor as capsaicin, and has been used medicinally as well as an insecticide (Szallasi, 2005).

As shown in Fig. 1, we were able to demonstrate that N2 significantly avoids piperine, at concentrations as low as 0.1%. We found especially interesting the wild-type response to piperine, which could be characterized as an “exaggerated backing” or “sneeze” response. Animals approach the piperine, withdraw with significant nose twitches/head shaking, and retreat quickly, often making at least one body length of backing, as shown in Fig. 2. They frequently repeat this behavior over minutes, to suggest that they are not adapting very quickly to the piperine.

We have also tested N2’s with allyl isothiocyanate and eugenol. (Allicin is highly unstable and not easily tested.) Worms appear not to avoid allyl isothiocyanate; however, they do robustly avoid eugenol—this latter result is in contradiction to previous results with this compound (Bargmann et al., 1993). Since eugenol is toxic to worms (Asha et al., 2001), it is reasonable to think that worms might avoid it, and we suspect that the previous result could have been due to the particular concentrations or conditions used.

In addition, we have begun testing the various TRPV mutants for their response to piperine. Since piperine functions on the same receptor as capsaicin, our prediction is that osm-9 and/or ocr-2 should show a specifically defective response.

We thank the CGC for providing strains.

Social interactions, learning experiences and responses to wide-ranging environmental stimuli occur through nervous cells via synapses. Several observations have suggested that the alteration of neuron connections during nervous system development could represent the root of many cases of autism spectrum disorder. Neuroligins are synaptic cell adhesion proteins that have been shown to be able to induce synaptogenesis. Single missense and frameshift mutations in neuroligin coding genes have been proposed that may lead to autism or mental retardation with complete penetrance (Baudouin and Scheiffele, 2010; Etherton et al., 2011). In C. elegans, nlg-1 is orthologous to mammal neuroligin genes, and it has been shown that nlg-1 mutants are defective in several sensory behaviors and sensitive to oxidative stress (Calahorro et al., 2009; Hunter et al., 2010).

We report here that transgenic expression of rat Nlgn1 and human NLGN1 proteins are functional in C. elegans. The wild type behavior pattern was rescued in nlg-1 deficient mutants by expressing cDNAs from rat Nlgn1 or human NLGN1 genes under C. elegans nlg-1 promoter (Figure 1A). Induced changes Asp396Stop in human NLGN1 or Arg453Cys in worm NLG1 proteins, failed to rescue the wild type phenotype (Figure 1B). These results indicate that mammalian and C. elegans neuroligins seem to be functionally comparable. The results anticipate that the nematode could be useful as an in vivo model for studying specific synapse mechanisms involved in autism. This system will allow the analysis of how mutations in neuroligin genes change phenotypes in different C. elegans genetic backgrounds and the study of their interactions with different environmental factors. It is probable also that this approach could be extended to other genes encoding synaptic proteins implicated in autism spectrum disorder, such as neurexins and shanks.

Table 1. Strains used in this study

^a Caenorhabditis Genetics Center.

^b Obtained by outcrossing VC228 strain with N2 six times.

^cThe cDNA nlg-1 coding region was obtained from clone yk1657a10, Yuji Kohara, National Institute of Genetics, Mishima, Japan.

^dThe cDNA human NLGN1 coding region was obtained from clone KIAA1070 (hj05602), Kazusa DNA Research Institute, Japan.

^e Rat Nlgn-1::EGFP was a gift from Dr. Thomas Dresbach, Univ. Göttingen, Germany.

The apparent simplicity and uniformity of the C. elegans nervous system belies a rich diversity of putative signaling molecules. In addition to the classical neurotransmitters, the C. elegans genome harbors a wide variety of bioactive peptides (flp, nlp and ins). Neuropeptides represent far and away the most abundant signaling molecules in the C. elegans nervous system and most of them are thought to function through the activation of G protein-coupled receptors. At the 18th International C. elegans Meeting, 2011, many presentations mentioned the involvement of neuropeptidergic signaling pathways in their field of research but hardly any data was presented on the biochemical coupling between the receptors and their peptide ligands. Despite the general knowledge and repeated predictions of peptide GPCRs following the elucidation of the C. elegans genome in 1998, only a handful of these have been deorphanized so far. Over the years, we have specialized in the identification of receptor-ligand couples and have developed an elegant way to maximize the chances of a successful receptor deorphanization using a combined reverse pharmacology approach.

First, sequence specific primers are designed and used to amplify, clone and verify the open reading frame of the GPCR of interest. The high-level eukaryotic expression vector pcDNA3.1^TM (Invitrogen) is used to generate high recombinant expression in mammalian cells driven by the human cytomegalovirus (CMV) promoter. This construct is then transiently expressed in Chinese Hamster Ovary (CHO-K1) cells and/or Human Embryonic Kidney (HEK293T) cells coexpressing the promiscuous G-alpha₁₆ subunit, which directs intracellular signaling to a calcium flux regardless of the endogenous G protein coupling of the receptor of interest. The resulting calcium flux upon receptor activation is monitored using the calcium sensitive photoprotein aequorin or the fluorescent calcium indicator Fluo-4 (Figure 1).

To identify the cognate ligand, we use a combination of three reverse pharmacological approaches: the tissue extract-based, the library-based and the information-based approach (Figure 2). For the tissue extract-based approach, a peptide extract from approximately 30,000,000 whole body mix stage worms was made and then subjected to reversed-phase HPLC fractionation. In addition to their direct use in the cellular pharmacological assays, the resulting peptide fractions were also studied in detail by mass spectrometric analysis (MALDI-TOFand ESI-Qq-TOFMS/MS Mass Spectrometry) to identify potential new peptides of interest. Based on bio-informatic predictions (“library-based approach”) and the mass spectrometric analysis of the reversed-phase HPLC fractions (“information-based approach”), a collection of 262 peptides, belonging to the established FLP and NLP families of peptides, was selected and custom synthesized. The peptides were selected in such a way that maximizes the number of different potential activating ligands in the combined library of synthetic and natural (endogenous) peptides (Figure 2). This combined approach enables us to test more than 262 putative peptide ligands of C. elegans for activation of orphan GPCRs.

This strategy has proven its worth as we have already uncovered more than a dozen C. elegans neuropeptide signaling systems in this way, including pigment dispersing factor (PDF), cholecystokinin (CCK) and gonadotropin releasing hormone (GnRH) signaling systems amongst others.

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The use of 2% agarose pads is the conventional current method of choice for nematode microscopy.  Although reliable, agarose pads are cumbersome to prepare and require the use of an immobilizing drug to keep nematodes motionless during the microscopy. Furthermore, a fast solidifying agarose drop on a glass slide requires some extra caution to make sure that the pad height remains minimum for microscopy with 40x or 100x lenses. We have developed a simpler and faster method for mounting nematodes for microscopy that does not require anesthetic drug. We report that a blend of equal percentage (20:20) of PEG 20000 and glycerol in 1x PBS provides enough viscosity to immobilize nematodes without distorting the cuticle/body shape. The treatment does not induce any observable osmotic shock when used on adult nematodes.

To perform the procedure, nematodes cleansed of bacteria are picked and then released into a PEG/glycerol droplet (6.5 μl) on a glass slide.  A cover slip is then gently placed on the droplet containing the nematode. Due to the small volume of PEG/glycerol droplet, nematodes need to be transferred to PEG/glycerol droplet, and rapidly overlaid with a cover slip before mounting medium (PEG/glycerol) dries. This quick step instantly immobilizes the nematode and leaves them well suited for microcopy, including acquiring images for extended periods of time. We find the method is particularly useful for quick screening of GFP expressing lines, which frequently requires analysis of many nematodes, and use of high-resolution, compound microscopy for detecting low levels of GFP expression (such as due to certain weak promoters).

We have also experimented with other mixtures of PEG and glycerol.  For instance, a mixture of 30% PEG 8000 in 25% glycerol also renders the nematodes amenable for microcopy, however increasing PEG concentrations above 30% renders nematodes unstable and unsuitable for microscopy.  Larval nematodes appear to be slightly more sensitive to the procedure.  We noticed slight cuticle distortion in younger nematodes (L1-L3) when PEG/Glycerol (20:20) solution was used for microscopy.  A further dilution of PEG might be required to overcome the distortion and 1 μl of levamisole (20mM stock) can be used to insure that the nematodes are motionless.

Developmentally arrested dauer stages in nematodes for laboratory assays have so far been obtained either by simply starving culture plates or by liquid culture in large Erlenmeyer flasks. Harvesting dauers from starved agar plates is straightforward but the yield is usually very low. Liquid cultures on the other hand produce high yields, but their maintenance is labor-intensive. We have combined the simple setup of starved plates with the increased yield of liquid cultures in the "wet plate protocol":

1. Wash 3 well-populated culture plates of worms into a centrifuge tube using sterile dH2O. Pellet down and remove supernatant.
2. Add 20ml of fresh E. coli OP50 grown in L-Broth medium.
3. To start the wet plates, use 9 freshly poured 10cm NGM plates. The plates should not have been dried after pouring.
4. Seed each plate with 2ml of worm/OP50 mixture.
5. Place the plates in a Tupperware^® box. Add moistened tissue to keep the interior of the boxes humid and incubate at 25° C.
6. Check the plates every second day to ensure conditions are still humid.
7. Depending on species, the plates are ready to harvest with lots of dauer larvae present after about 10 days.

It is important to maintain a layer of liquid on the plates at all times. If the worms are able to crawl instead of swim, the plates are already too dry. Just like liquid cultures in an Erlenmeyer flask, liquid plates may become contaminated. It is therefore important to prepare the plates to be as sterile as possible (i.e. under a flowhood). If problems persist, the culture may either be started with freshly bleached eggs instead of a mixed culture. Additionally, antibiotics and fungicides may be added to the worm/OP50 mixture.

For large-scale experiments that require huge numbers of dauers (e.g. microarrays), the use of liquid cultures is preferable. For smaller experiments however, the wet plate protocol has proven to be simpler, quicker and less prone to contamination in many assays in our lab.

The Notch-like receptor GLP-1 is defined by several temperature sensitive and non-conditional alleles. The glp-1 alleles e2141 and e2144 were isolated by Ralf and Heinke Schnabel in a temperature-dependent embryonic lethal screen at the MRC, Cambridge. Our results suggest that there was a mix-up in the distribution or propagation of the e2141 allele, such that the "e2141" allele that the Priess lab has maintained and subsequently distributed appears to be identical to e2144. The Priess lab apologizes for any inconvenience associated with previous distribution of "e2141". In short, contrary to what was previously thought and published (Kodoyianni et al., 1992 and also in WormBase), e2141 and e2144 do not bear the same sequence change. e2141 actually carries two mutations in exon 8: c2920t and a3610g.

We urge the community to sequence their "e2141"-bearing lab strains prior to future publication or dissemination. We note that in the prior literature, strains designated as "e2141" may have been carrying the e2144 (c2785t) mutation. In light of our sequencing results, the literature indicates that the e2144 (c2785t mutation variously attributed to "e2141" or "e2144") results in a much stronger embryonic phenotype than e2141 (c2920t a3610g mutations). See, for example, Mello et al., 1994, and Hutter and Schnabel, 1994. The e2141 allele is in the CGC collection as strain CB4037, and the e2144 allele is now deposited at the CGC as strain GE68.

RRF-3 is one of the four putative RNA-directed RNA polymerases (RdRPs) in C. elegans and plays a major role in endogenous synthesis of siRNAs. By deep sequencing of mRNA tags (mRNA-seq) from N2 worms and from worms with mutational backgrounds, at different stages, we found that the annotated seventh exon of rrf-3 contains an intron. The position of the intron is chrII: 8165255-8165300 (WS190 version of the C. elegans genome), and it is 45 bp long (Figure 1). The 5' end of the intron starts with GTATATTCTA and the 3' ends with AAATATTCAG. The intron does not change the reading frame and shortens the protein by 15 amino acids.

We recently acquired a COPAS worm sorter and found that the analysis software included could not do what we needed. For this reason, we began developing a framework tailored to analyze the data. So far we have developed the functionality to produce chronograms (Dupuy et al., 2007) and to compare subregions statistically. The scatter plots produced are easy to navigate and can be overlaid to compare different runs.

The software is open source, enabling anyone to extend or modify it to fit their purpose. It is split into two parts: a library that can be used within your own code (e.g. for high-throughput analysis) and a graphical user interface for manual visualization and analysis of the data. All code is written in Java.

The code is already available at jflux.sourceforge.net, but the software is still in early development. We hope that other COPAS users are interested in contributing to this project. Having an extensible platform will allow us to incorporate new analysis methods to make the most out of the data.

We have generated an extensive library of C. elegans promoters as Invitrogen Gateway entry clones to facilitate easy cloning of tissue-specific reporters. As a lab that focuses on males, we have analyzed the expression of many of the promoters in the male. In addition to an extensive list of published promoters (Table 1) (Gruninger et al., 2006; Gruninger et al., 2008; LeBoeuf et al., 2007; Liu et al., 2007; Reiner et al., 2006), we have a large library of promoters in entry clones that have not been published (Table 2), and we wish to make these plasmids available to the worm community. We have also included a list of Gateway destination vectors we have generated (Table 3). Plasmids available are listed in the tables below.

Contact Dr. Rene Garcia to obtain plasmids of interest.

To study the expression pattern of innexins in the male worm, we crossed innexin reporter strains (Altun et al., 2009) with him-5(e1490) strains and analyzed GFP expression using a compound microscope. As describe in the table, innexins are expressed in different tissues and cells of the male worm. These results will be compared to the male connectome project where a lot of gap junctions have been observed in the nervous system.

The C. elegans hermaphrodite gonad is formed by migration of the two distal tip cells (DTC). These cells migrate away from the gonad primordium on the ventral side of the animal (one anterior and the other posterior), turn dorsally and migrate back toward the center of the animal on the dorsal surface resulting in two U-shaped gonad arms of proliferating germ cells (Kimble and White, 1981). We are interested in genes that affect this migratory path and performed a genome-wide RNA interference (RNAi) screen that revealed 99 genes required for DTC migration (Cram et al., 2006). In this screen, the RNAi-sensitized strain rrf-3(pk1426) was subjected to RNAi by feeding. In this approach, all tissues have the potential to be affected by RNAi; therefore DTC migration might be affected by RNAi knockdown in tissues other than the gonad. To overcome this, we took advantage of the lag-2p::GFP; rde-1(ne219); lag-2p::rde-1 strain of C. elegans (generated by Dr. Dana Byrd and Dr. Judith Kimble) that allows RNAi to have an effect mainly in the two DTCs.

We rescreened the 99 genes required for DTC migration with the lag-2p::GFP; rde-1(ne219); lag-2p::rde-1 strain to identify genes that act cell autonomously. We applied the same two step microscopy approach as Cram et al. (2006), first looking under light microscopy with a dissecting microscope and scoring for worms that show clear patches (intestinal displacement due to gonad abnormal migration or distension). In the second step, we characterized the migratory paths using DIC microscopy and put them into categories (types 1-3) based on gonad morphology. P0 and F1 generations were scored in both screens. The use of the rrf-3(pk1426) sensitized strain resulted in a higher percentage of animals with clear patches in both generations in the primary screen compared to the lag-2p::GFP; rde-1(ne219); lag-2p::rde-1 strain. Of those adults with clear patches that were analyzed in the secondary screen, the proportions of the three types of DTC migration defects were similar between the two strains. Therefore, whole animal and DTC-specific RNAi give similar DTC migration phenotypes but with different penetrance.

Knockdown of 28 of the 99 genes caused DTC migratory defects in 30% or more of the hermaphrodites. Another 31 genes affected DTC migration in 5-29% of adults. Thus, we conclude that these 59 genes have cell autonomous activities during DTC migration. No significant effect on DTC migration was observed in the remaining 40 genes. However, we cannot conclude that these do not act cell autonomously because lack of a defect could result from the less efficient knockdown in the DTC-specific strain. RNAi data are available at www.cellmigration.org/resource/discovery/schwarzbauer/schwarzbauer_dtc_auto.cgi.

In the study of C. elegans navigational behaviors, it helps to be able to quantify worm movements on spatial gradients that can be manufactured with precision, speed, and ease. It has been straightforward to produce such gradients with stimuli like temperature (Ryu and Samuel, 2002), but less so with chemical stimuli (Pierce-Shimomura et al., 1999; Iino and Yoshida, 2009). Here, we describe a simple way to make linear gradients of soluble chemicals for studies of worm chemotaxis.

1. Elevate one side of a square Petri plate as shown, such that the bottom of one side is level with the top of the opposite side (Fig. 1a).
2. Fill with melted agar with a chemical concentration a, and wait until it hardens, creating a triangular wedge of agar (Fig. 1b).
3. Lay the plate flat. Now fill with melted agar with a chemical concentration b, and wait until it hardens. Agar hardens at a lower temperature (<30°C) than it melts (>60°C), so the first triangular wedge is not disrupted as the second wedge is poured (Fig. 1c).

Diffusion will first equilibrate the soluble chemical in the vertical direction (Fig. 1d). The diffusion equation, x² = 2Dt, tells us how long we need to wait. Using the diffusion coefficient for a small molecule in water (D = 10^-5 cm² s^-1), one estimates that the molecules diffuse ~1 cm in ~12 hr. So, after ~1 day, the surface of the agar plate will have a linearly varying concentration of chemoattractant, a on one side, b on the other. The diffusion equation also tells us when the gradient disappears in the horizontal direction (after ~40 days with 9 cm plates).

A tiny amount of methylene blue in one of the agar solutions helps to visualize the gradient. Fig. 2a shows a photograph of the edge of a 24 cm x 24 cm agar plate shortly after the second wedge was poured. Fig 2b is a photograph of the surface of the same plate taken the following day.

We have used this linear gradient assay to study chemotaxis towards NaCl. We use a high-resolution tracking system to follow the movements of individual worms navigating linear gradients on 24 cm x 24 cm square agar plates. Each panel of Fig. 3 shows each track of individual wild-type worms navigating linear NaCl gradients spanning 0 to 50 mM (Fig. 3a) or 50 to 100 mM (Fig. 3b, c).  For presentation purposes, all tracks in each experiment are depicted as starting from a common point (red circles in each panel). Worms grown on standard NGM plates (50 mM NaCl) will crawl towards 50 mM NaCl whether initially placed at 25 mM NaCl (Fig. 3a) or at 75 mM NaCl (Fig. 3b). On the other hand, worms grown on high salt NGM plates (100 mM NaCl) will crawl towards 100 mM NaCl (Fig. 3c). Thus, worms develop a preference for the salt concentration that corresponds to their cultivation conditions, and are capable of either moving up gradients or down gradients in pursuit of their preferred salt concentration.

For us, the linear gradient assay has opened new doors in the study of the different navigational modes in NaCl chemotaxis (positive chemotaxis up gradients, negative chemotaxis down gradients) as well as experience-dependent plasticity. As any soluble chemical can be used, the method should facilitate numerous studies in C. elegans chemotaxis.

C. elegans is known to be attracted to various chemicals including volatile odorants and water-soluble salts. Chemotaxis to salts is conventionally tested on agar plates with salt concentration gradient. Most popular ways to make the gradient plate is either: 1) to spot a drop of concentrated salt on a chemotaxis plate (either once or twice at separate time points) and allow diffusion for several hours to overnight (Ward, 1973; Iino and Yoshida, 2009), or 2) to place a small agar block containing high concentration of salt on a chemotaxis plate and allow diffusion (Tomioka et al., 2006). Both of these procedures lead to concentration gradient ranging from, say, 10-20 mM at the peak to several millimolars at the periphery, corresponding to the salt concentration of basal buffer in the chemotaxis agar. The standard procedure therefore tests the worm’s behavior only in this low salt concentration range.

Here we show that by simply including salt in the background agar, chemotaxis at a much broader range of salt concentration can be tested. In Figure 1, background agar includes 25 mM potassium phosphate (pH 6.0), 1 mM CaCl₂, 1 mM MgSO₄, and 50 mM NaCl. On top of the agar plate with this composition, two cylindrical agar blocks were placed (each 15 mm in diameter and 5 mm in thickness, excised from an agar plate using a cork borer) that included either 0 mM (left side) or 150 mM (right side) of NaCl in addition to the chemotaxis buffer for 16 hours. This allows creating a concentric salt gradient on the test plate with highest concentration at the center of the high salt plug, lowest at the opposite side, and background concentration around the center. Animals washed from culture plates were then placed off center on the test plates to observe chemotaxis.

One remarkable observation that came out of this assay is that worms are not merely attracted to salt, but they are attracted to the salt concentration at which they have been raised. In addition, when worms are starved at various salt concentrations and then tested on the same assay plates, they now avoid the salt concentration that they have experienced. Therefore, the behavior on salt concentration gradient is based on salt concentration memory and depends on the food availability they experienced (a brief report on this observation was presented at the EAWM, 2010). This manner of behavioral control is reminiscent of that already known for thermotaxis, except that we have not so far observed obvious tracks along the line with same salt concentration.

C. elegans can recognize different environments by using chemical signatures from environmental stimuli or from other worms. We investigated whether C. elegans are able to recognize themselves by testing whether they could discriminate between unexplored environments, environments they themselves previously explored, or environments previously explored by a conspecific.

To ensure that most, if not all, of the E. coli were removed from worms they were each placed in a drop of M9 buffer for 5 minutes on a blank NGM plate then placed out of the drop on the plate for an additional 5 minutes prior to the trial. Following washing, single worms were placed on a fresh NGM agar plate for an initial 30 minute observation period and then either lifted with a platinum pick and put back onto their original plate (sham switch), lifted and moved to an unexplored NGM plate, or lifted and moved to an NGM plate previously explored by a single conspecific for 30 min. The time the worms were lifted off the plates was held constant between the groups.

We measured and compared the speed of the worms at the beginning (min 2-5) and end (min 27-30) of the 30 min exploration of the first plate and at the beginning of the time on the subsequent plate (min 32-35). Speed was measured as a proxy for foraging behavior (Bargmann, 2006). Rate of spontaneous reversals and foraging range were found to be insufficiently robust to be suitable measures. For statistical analyses ANOVAs were used to compare the speeds of locomotion between the beginning of the first 30 min (blue bars) to the average speed at end of the first 30 min (red bars), and the average speed at min 32-35 (min 2-5 of the second 30 min period; green bars) for each of the three groups. A Bonferroni correction for multiple tests set significance at p< 0.016.

The first minute after each transfer (to the initial plate and to the new plate after 30 min) all worms moved rapidly (data not shown), therefore the first two minutes of each 30 min period were not included in our analysis. There was no change in the rate of speed from min 2-5 to min 27-30 of the first 30 min observation period for any of the three conditions (Fig 1).  ANOVAs for the sham switch group and for the group switched to a plate previously explored by another worm showed no significant differences in speed between the three timepoints tested.  In contrast, an ANOVA for the group switched to a blank unexplored plate was significant (p<0.000044): post-hoc tests indicated that the their speed in min 32-35 (min 2-5 of the second observation period) was significantly slower than their speed at either min 2-5 or min 27-30 of the first observation period.  Thus C. elegans moved significantly more slowly on a plate that had not been explored by a worm (either themselves or a conspecific) than on a previously explored plate.

Although it is possible that the explored plates might have had trace amounts of E. coli it is less likely than the hypothesis that some worm pheromone allowed worms to distinguish plates that had contained worms from plates that had not. Worm tracks from each of the three groups were very similar and did not indicate that worms on a blank plate explored any differently that worms in the two "previously explored plate" conditions.  Unexplored plates elicited slower movement than plates containing worm odor. Taken together the data suggest that C. elegans do not have the ability to distinguish self from conspecifics (and pilot data suggests self from C. briggsae), but do distinguish between environments where worms have been and environments where there have been no worms.

It is known that sensory information about relatively constant environmental factors plays important role in formation of relatively constant functional states of human and mammalian nervous system. Therefore strong decrease of total volume of sensory information (sensory deprivation) causes disintegration of normal human and rodents' behavior.

In order to investigate possible influence of total volume of sensory information on the functional state of C. elegans nervous system young adults, grown in Petri dishes with NGM and E. coli OP50 (Brenner, 1974) at 18°C, were incubated individually in 1 ml of liquid medium (NGM without agar, peptone and cholesterol) during 0.5, 1, 2 or 3 hours. In these conditions sensory deprivation of worms is determined by cessation of sensory inputs from conspecific individuals, food and agar plate. Our experiments show that sensory deprivation rapidly (30 min at 18°C) diminished spontaneous swimming rate and then completely stopped worms' swimming (after 90 min incubation). This effect of sensory deprivation is prevented by sensory inputs from conspecific worms, since about 85% of worms maintain spontaneous swimming after 3 hours incubation of 100-200 worms in 3 ml of liquid medium. Temperature increase from 18 to 25°C strongly stabilized worms swimming in conditions of sensory deprivation.

It is known that worms' pretreatment by serotonin (4 or 12 hours) causes adaptation of C. elegans serotonin receptors (Schafer and Kenyon, 1995). This adaptation reveals in the absence of behavior response to exogenous serotonin in fresh medium (Schafer and Kenyon, 1995). In our experiments such C. elegans adaptation to serotonin (12 hours on agar plates containing E. coli OP50 and serotonin 3 mg/ml) diminished effect of sensory deprivation at 18°C, but not at 25°C.

Since exogenous serotonin inhibits C. elegans movement, influence of C. elegans adaptation to serotonin on effect of sensory deprivation displays possible role of endogenous serotonin in this effect.

In whole our data indicate that sensory deprivation can be used for investigation of C. elegans nervous system regulation by continuous and relatively constant sensory inputs, such as social signals and stimulation of inadaptable or partly adaptable thermosensory neurons by constant environmental temperature.

The nematode Caenorhabditis elegans, with information on neural connectivity, three-dimensional position and cell lineage, provides a unique system for understanding the development of neural networks. Although C. elegans has been widely studied in the past, we present the first statistical study from a developmental perspective, with findings that raise interesting suggestions on the establishment of long-distance connections and network hubs. Here, we analyze the neuro-development for temporal and spatial features, using birth times of neurons and their three-dimensional positions. Comparisons of growth in C. elegans with random spatial network growth highlight two findings relevant to neural network development.

First, most neurons which are linked by long-distance connections are born around the same time and early on, suggesting the possibility of early contact or interaction between connected neurons during development. Second, early-born neurons are more highly connected (tendency to form hubs) than later born neurons. This indicates that the longer time frame available to them might underlie high connectivity. Both outcomes are not observed for random connection formation. The study finds that around one-third of electrically coupled long-range connections are late forming, raising the question of what mechanisms are involved in ensuring their accuracy, particularly in light of the extremely invariant connectivity observed in C. elegans.

In conclusion, the sequence of neural network development highlights the possibility of early contact or interaction in securing long-distance and high-degree connectivity.

The article reporting these results is currently in press (Varier and Kaiser, 2010). Earlier work on C. elegans (Kaiser and Hilgetag, 2006), neural network development (Kaiser et al., 2009), and neural network analysis (Kaiser, 2007) is listed below for further reference. Both the current article and the data sets showing the network at different developmental stages will be made available on www.biological-networks.org upon publication (within the next couple of weeks).

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C. elegans transgenes are usually maintained as unstable extrachromosomal arrays, requiring the manual picking of animals using visible markers. This can be laborious and limiting for some assays. To overcome these shortcomings, our two groups independently developed methods for using antibiotic selection in C. elegans. We developed nematode transformation vectors carrying antibiotic resistance genes (Neomycin and Puromycin, respectively) and found that they could be used as very efficient selection markers. Selection can be performed in both liquid media and on agar plates, and has no obvious effect on phenotype. The two selection systems allow hands-off maintenance and enrichment of non-integrated transgenic animals, as well as isolation of MosSCI-generated single copy integrants. No particular genetic background is needed and both selection protocols also work in C. briggsae. We hope therefore that the approach will be useful to many worm biologists, including those working on species other than C. elegans. To our knowledge this is the first time that antibiotic resistance markers have been successfully used in whole animals, and so the approach may open up opportunities for research using other ‘non-model’ organisms.

Biolistic transformation is currently a popular method for making trangenic lines in C. elegans. Although this method is reliable, the fraction of successfully transformed animals that is obtained is extremely small, necessitating the use of a co-transformation marker. The most commonly used marker is the unc-119(+) gene which can dominantly rescue the severe Unc and dauer defective phenotypes of unc-119(ed3) mutants. Despite the utility of the unc-119(+) marker, screening for rare transgenic animals amongst the progeny of bombarded animals is time consuming and labor intensive. One approach is to allow the plates containing the bombarded animals to starve over the course of a few weeks. Each plate is then divided into chunks and the chunks moved to fresh plates containing food. These plates can then be screened a day or so later for animals with normal mobility. This approach is still time consuming and has the added drawback of amplifying the number of plates (and therefore the surface area) that has to be scanned. I have devised a simple alternative to this approach in which the worms do most of the work. One to two weeks after bombardment, just after the plates have starved, a fresh plate containing OP50 is cut up into little (1-2 cm²) chunks. Each of these chunks is placed on one of the plates containing the bombarded animals so that the side containing the bacteria is face up (away from the worms). The plates are left upright in the incubator overnight and the next day the bacterial lawn on top of the transferred chuck is checked for rescued worms. Because the animals have to climb up to the top of the chuck to reach the food, the rescued animals have a significant advantage. Using this approach, my group has on multiple occasions, been able to identify a single transformed worm from a 100 mm plate. This approach has the added advantage of being rapid; rather than scouring the entire plate for transgenic worms, only the small area on top of these "pedestals of rescue" has to be searched.

Two of our methods are now seeing publication. We are writing to let people know how to get the relevant strains and constructs. The first method uses the ninth intron of mec-2, whose splicing depends on mec-8, to produce temperature-sensitive expression and temperature-sensitive RNAi (Calixto et al., 2010a). The intron 9 vector TU#821 (P_mec-18 intron-9 mec-2::yfp) is available from Addgene (www.addgene.org/Martin_Chalfie). The TU218 [mec-8(u218ts)] and TU3135 [mec-8(u218); rde-1(ne219); uIs46 (ceh-22::GFP, P_rde-1mec-2 intron9::rde-1)] strains are available from the C. elegans Genetic Center (CGC).

The second method uses SID-1 expression in neurons to allow feeding RNAi for neuronally-expressed genes (Calixto et al., 2010b). Expression of neuronal sid-1 in the RNAi sensitized strain lin-15b(n744) greatly enhanced this effect. Furthermore, promoter-driven sid-1(+) can be used with a sid-1 mutant background to restrict RNAi to specific cells. Currently we are using neuronal and touch receptor neuron-specific expression of sid-1 to uncover neuronal and touch neuron-specific defects for genes whose loss has more general effects (e.g. lethality). The following strains have been submitted to the CGC. In addition, a vector allowing pan-neuronal sid-1 expression TU#867 (P_unc-119 sid-1) will be available from Addgene.

The adherence of worms to standard plastic tips makes accurate dispensing difficult. While using glass tips can overcome this problem, their application is limited when sterility and frequent tip changing is needed, for example, when performing liquid culture RNAi screens using 96-well plates (Lehner et al., 2006). RNAi screen protocols often require ~10 L1 worms to be dispensed into each well of a 96 well plate, however using standard P200 tips with worms in M9 alone resulted in an average variance of ± 6 L1 worms per well. We found that worms suspended in M9 with Triton X-100 decreased this adherence, resulting in an average variance of ± 2 L1 worms in each 10µl volume.

Both Triton X-100 and Tween-20 can be used to decrease this adherence to standard P200 tips. Since autoclaving either of these reagents is not recommended, sterilization can be carried out via filtration using a 2µm filter. The lower viscosity of Triton X-100 over Tween-20 greatly aided filtration and sterility tests showed no associated contamination. We found that using concentrations of Triton X in M9 ranging from 0.1% to as low as 0.01% proved to be effective in decreasing worm adherence throughout our assay. No negative issues regarding the growth or progeny of worms were observed when compared to a control plate containing no Triton X. Therefore this method could serve useful when applied to protocols that require consistently small numbers of worms in large repetitions.