Hill DP et al. (1986) Worm Breeder's Gazette "the critical times microfilaments are needed to generate asymmetries in the first cell division of C elegans."

Strome S, Hill DP

[Worm Breeder's Gazette, 1986]

Recently, we have been determining the time microfilaments (MFs) are needed to generate and maintain proper asymmetry during the first cell cycle of Caenorhabditis We can disrupt the MF system using the MF inhibitor cytochalasin D (CD). As an indication of asymmetry, we have been observing the behaviour of the zygote during pseudocleavage, pronuclear migration, segregation of germ-cell- specific granules (P granules), and first cell cleavage, all of which clearly show some indication of asymmetry. As has already been reported, when embryos are made permeable to CD early in the first cell cycle all manifestations of asymmetry are destroyed: pseudocleavage disappears, the egg and sperm yronuclei meet in the center of the embryo, P granules fail to segregate posteriorly and the first mitotic spindle sets up and remains symmetrically placed throughout karyokinesis. If one-cell embryos are treated with CD after the pronuclei have met and P granules have been segragated, P granules remain posterior but the mitotic spindle sets up and remains symmetric. This suggests that CD affects those events which have not yet taken place, but does not reverse the asymmetries already generated. It also suggests that MFs are not required to hold P granules in the posterior of the embryo once they have been segregated there. They may be anchored to non-MF components or may not be free to diffuse due to the nature of the cytoplasm. Zygotes can be pulsed with CD by introducing drug and then washing the drug out of the zygotes at specific times. Control experiments show that CD disrupts MFs within one minute of drug treatment. The minimum time zygotes were treated with drug during experiments was two minutes. Based on three types of CD pulse experiments (see diagram), it appears that MFs are not required prior to pseudocleavage and pronuclear migration but are required from pronuclear meeting until cleavage to generate proper asymmetry. l)If the embryo is treated very early, during pronuclear formation or early pseudocleavage, and the drug is washed out two minutes later, the embryo will resume pseudocleaving and behave like a normal embryo with respect to all observable aspects of asymmetry. 2)If CD is added to the embryo prior to pronuclear migration and washed out shortly after the pronuclei reach the center of the embryo, the embryo is able to recover enough to form a cleavage furrow and carry out cytokinesis. However, the proper asymmetry of the cleavage is lost. Most often the embryo divides into two equal-sized daughters, although a small percentage of the time the furrow is formed to give either a slightly larger AB-cell or a slightly larger Pcell. The P granules are not able to recover from the treatment and remain localized in the center of the embryo as they do in a continuously cytochalasin-treated embryo. 3) Preliminary results show that embryos treated shortly after pronuclear migration and P granule segregation and then washed several minutes later also undergo symmetric cleavage. However, in these embryos, P granules are segregated to the posterior cell. We conclude from these experiments that proper MF structure is needed at a critical time, during late pseudocleavage and pronuclear migration, in order for pronuclei to meet properly and P granules to segregate properly. After this time, microfilaments are needed in order to generate proper asymmetry in the first cell division itself but are dispensible with respect to events which have already occurred (P granule localization). Further experiments will be done with various cytochalasin-pulsed embryos to examine how pulses affect the developmental potential of daughter cells. {Figure 1}

Waterston RH (1977) Worm Breeder's Gazette "R73 - a correction."

Waterston RH

[Worm Breeder's Gazette, 1977]

The strain R73 has previously been described as a revertant of unc- 54(e569) (Epstein, et al., J. Mol. Biol. (1974) 90:291-300; Mackenzie and Epstein, Nematode workshop, 1977). However, the fine structure as described by Mackenzie in Woods Hole strongly resembled the muscle of putative missense alleles of unc-15 (Waterston, et al., J. Mol. Biol. in press) and not that of any of the other mutants so far isolated and examined (Waterston, unpublished). Accordingly, R73 was obtained from H. Epstein and further genetic studies carried out. R73 fails to complement unc-15(e1214), with R73/e1214 heterozygotes resembling R73 in their muscle structure by polarizing microscopy. This defect in muscle structure maps close to dpy-5 I, and three factor crosses indicate that the mutation(s) lies near or to the right of unc-13. No evidence for a mutation(s) in the unc-54 gene was found. Thus R73 is apparently a partial revertant of an unc-15 allele, probably e73 and not unc-54(e569) (Where the error in bookkeeping occurred is unclear), and the large paracrystalline arrays, presumably of paramyosin, remain characteristic only of presumptive missense alleles of unc-15.

Hill DP et al. (1987) Worm Breeder's Gazette "developmental consequences of brief microfilament disruption in the first cell cycle of C elegans."

Strome S, Hill DP

[Worm Breeder's Gazette, 1987]

We are continuing the studies that we reported at the worm meeting by analyzing the fates of embryos that have been pulsed with cytochalasin D (CD) in the first cell cycle. We previously showed that if we pulse embryos with CD during pronuclear migration and the later stages of pseudocleavage we disrupt many of the aspects of asymmetry that we monitor: pseudocleavage, the posterior meeting of the pronuclei, segregation of germ-line specific P granules, posterior positioning of the mitotic spindle, and the generation of a large AB cell and a smaller P cell. Disruption of microfilaments before or after this 'critical interval' seems to have no effect on the formation of a normal two-cell. To analyze the developmental consequences of brief microfilament disruption during the 1-cell stage, we've been assaying 1) the early cleavage patterns and cell cycle rates of pulsed embryos, 2) the internal migration of the germ-line cells, Z2 and Z3, as an indication of gastrulation, 3) the appearance of three differentiation markers: gut granules, paramyosin and a seam cell antigen, and 4) morphogenesis.

Schachat FH et al. (1978) Worm Breeder's Gazette "Myosins exist as homodimers of heavy chains: Demonstration with specific antibody purified by nematode mutant myosin affinity chromatography."

Epstein HF, Schachat FH, Garcea RL

[Worm Breeder's Gazette, 1978]

The body-walls of Caenorhabditis two different myosin heavy chains that associate to form at least two species of myosin. In order to better define the distribution of these heavy chains in myosin molecules, we have characterized the myosin of C. elegans by immunochemical methods. Specific, precipitating anti-myosin antibody has been prepared in rabbits using highly purified nematode myosin as the immunogen. The difference in reactivity of the anti-myosin antibody with wild-type myosin containing both kinds of heavy chains (designated unc-54 and non-unc- 54 heavy chains on the basis of genetic specification) and myosin from the mutant E190 that lacks unc-54 heavy chain indicates that there are antigenic differences between myosin molecules containing unc-54 heavy chains and myosin molecules containing only non-unc-54 myosin heavy chains. Antibody specific for the unc-54 myosin determinants has been prepared by the immunoadsorption of anti-myosin antibody with E190 myosin. This specific antiunc-54 myosin antibody precipitates myosin that contains only unc-54 heavy chains. At the limits of resolution of our immunoprecipitation techniques, no heterodimeric myosin molecules that contain both unc-54 and non-unc-54 heavy chains can be detected. Therefore, the body-wall myosins of C. elegans exist only as homodimers of either class of heavy chain. This specific anti-unc-54 myosin antibody promises to be a valuable tool in understanding the role of two myosins in body-wall muscle and in molecular characterizations of mutant myosins in C. elegans. We report here the use of this antibody to detect antigenic differences between unc-54 myosin from the wild-type and the muscle mutant E675. In conjunction with the original anti-myosin antibody, other studies show that both unc-54 and non-unc-54 myosins exist within the same body-wall muscle cells (See Mackenzie, Schachat and Epstein) and that both myosins are coordinately synthesized during muscle development in C. elegans (See Garcea, Schachat and Epstein).

Wilson SL et al. (1984) Worm Breeder's Gazette "an unc-52 mutant with temperature-dependent paralysis phenotype."

Wilson SL, Jacobson LA

[Worm Breeder's Gazette, 1984]

The unc-52 allele su250 was described by Mackenzie et al. [Cell 15:751-762 (1978)] as a derivative of the e669 allele which, when homozygous, produced only a mildly uncoordinated phenotype as compared with the total paralysis produced by allele e669. Both sarcomere organization and myosin heavy chain B accumulation in su250 homozygotes were reported to be intermediate between wild-type and animals homozygous for more severe unc-52 alleles [Zengel & Epstein, PNAS 77:852-856 (1980)]. In our hands, strain HE250 unc-52(su250)II, obtained from CGC, shows a temperature-dependent paralyzed phenotype. When grown at 16 C, the worms are nearly wild-type in size and movement rate, paralysis sets in late in adulthood (late egg-laying uncoordination sets in earlier, becoming complete paralysis around the beginning of egg-laying. Worms grown at 20 C show an intermediate phenotype: partial paralysis is evident by early egg-laying and total paralysis occurs only late in adulthood. Outcross results with HE250 indicate that the ts phenotype does not result from extragenic suppression. Furthermore, when su250 is placed over an unc-52 null (e669) or a deficiency (jDf02), the animals are completely paralyzed even at 16 C. We infer that a single copy of the su250 allele produces insufficient functional gene product to sustain movement. Temperature shift experiments indicate that muscle function, rather than muscle morphogenesis alone, may be temperature-dependent. Adult su250 homozygotes grown at 16 C become noticeably impaired within 8 hrs. after an upshift to 25 C, and totally paralyzed in less than 240 hrs. Conversely, larvae or adults downshifted from 25 C to 16 C retain whatever degree of motor impairment they had at the time of the downshift. Thus, the phenotypic effect of high temperature is not reversible. We had previously reported (Worm Meetings, 1983) that unc-52 mutants are deficient in lysosomal cathepsin D. Both enzyme activity and cathepsin D protein (measured by quantitative immunoblots) are near- normal (50-75% of wt) in HE250 grown at 16 C, and are reduced (15-20% of wt) in paralyzed HE250 grown at 25 C. This matter is still under investigation, but we believe now that the cathepsin D deficiency is an indirect effect of underfeeding in paralyzed animals (see Clokey & Jacobson, this issue).

Donkin SG et al. (1993) Worm Breeder's Gazette "C. elegans as a Soil Toxicity Test Organism"

Donkin SG, Dusenbery DB

[Worm Breeder's Gazette, 1993]

We have developed a new procedure for recovering nematodes from soils in an efficient and non-destructive manner, which has permitted the development of a short-term soil toxicity test using nematodes as toxicity indicators. The procedure involves gentle centrifugation through a colloidal silica suspension (Ludox AM or Ludox HS-40, both available from E. I. Du Pont de Nemours & Co., Wilmington, DE), which allows the soil to settle out while floating the nematodes on top of the suspension. The nematodes are recovered with great efficiency (~90%) and are unharmed by the recovery process. Short-term lethality tests (24-hour exposure) were performed with several metals (Cd, Cu, Hg, Ni, Pb, and Zn) in several different characterized soils, and replicable results were obtained, allowing concentration-response curves to be generated and LC(50)s to be estimated. It was found that the presence of soil generally decreased the toxicity of the metals to nematodes when compared to tests done without soil present. This is presumably due to the sorption of the metal ions to the soil particles, which lowered their availability to the nematodes. Comparison between nematode lethality results and data published for earthworms exposed to metals in soil revealed that, in most cases, the nematode is at least as sensitive as the earthworm in terms of its response to toxic metals in soil. The earthworm test, which is currently the standard animal soil toxicity test, has the disadvantage of taking two weeks to perform, while the nematode test can be done in 24 hours. Some correlations between soil or metal properties and the resulting lethal effects were obtained, while other properties showed no significant correlation with toxic effects. This suggests that, as other studies have indicated, the bioavailability of metals in soils cannot be reliably predicted by considering only a few sample conditions. Soils are complex and heterogeneous systems, and there are many physico-chemical processes that may occur within them and which may influence the resulting bioavailability of a contaminant. The C. elegans soil toxicity test may provide the means to identify some of the more important environmental characteristics that influence contaminant bioavailability.

Schatz BR et al. (1992) Worm Breeder's Gazette "Availability of WCS (Worm Community System), Release 1"

Ward S, Yeatts A, Friedman T, Hudson SJ, Peterson L, Grossman E, Powell K, Schatz BR

[Worm Breeder's Gazette, 1992]

Release 1 of WCS is now running in more than 10 worm labs. The goal of this system is to make "all" knowledge about C. elegans, both formal and informal, easily available to you in your labs. The intention of this release is to indicate possible functionality and to learn some of the major problems in providing a complete system useful to the community. While this "consciousness-raising" is on-going, we are actively designing Release 2. Starting next summer, our plan is to release and evolve a system of increasing functionality. We also plan to expand our project to provide active user support. Please send us your comments (to wcs@csl.biosci.arizona.edu) so that the system can become more useful to the community. Many thanks go to the people who have already used the early release and provided good feedback. WCS supports browsing multiple interconnected sources and adding new data. All data distribution is done automatically and electronically, both for the system-added archival sources, such as the WBG, and the user-added informal sources, such as annotations. The current sources include: the complete WBG, the CGC bibliography with abstracts where available (currently about half), abstracts from the last Worm Meeting, the gene list, the physical map, Genbank C. elegans sequences, CGC strains, WBG subscribers, and sample informal data. Thanks to Mark Edgley for help of many sorts. acedb (Durbin & Thierry-Mieg, WBG v12 ,n1 ,p13 )is distributed with WCS and runs side-by-side with it. The programs communicate, so that the acedb genetic map can be called from within WCS and a WCS annotation can be made from within acedb. Our plans for release 2 of WCS are to completely rewrite the current system, improving many inadequacies. Features being considered include: user entry of many types of data, e.g. gene descriptions, with appropriate editorial control; increased literature coverage with faster and more powerful search; portability across many common computer platforms; and a distributed architecture, with a separate front end and back end permitting multiple access. We are also investigating displays for additional worm data, e.g. the cell lineage, the cell anatomy, and the wiring diagram. In this regard, we are actively seeking a biologist to come work with us (see ad elsewhere in this issue). The current release runs on Sun workstations, although the display can be run on any X-windows terminal. A full setup, such as at Arizona, has a Macintosh in the worm lab running MacX connected via a building Ethernet to a central Sun file server running WCS. Note this requires an Ethernet card for the Mac, and preferably, a 2 page monitor. Most of the current sites have bought Sun workstations to run this software. One typical configuration is a SPARCstation-2 which for about $9000 university price comes with a monochrome monitor, 32 MB RAM, and 400 MB disk. Many sites have added an external 1 GB disk and additional 32 MB RAM for a $13K total cost. For instructions to obtain WCS, use ftp from a machine connected to the Internet: $ ftp csl.biosci.arizona.edu (ftp 128.196.124.17) Name: anonymous Password: your-login@your-machine ftp> cd wcs ftp> get README.FTP ftp> quit You can also run WCS remotely across Internet from your workstation running X-windows to our server by telnet csl.biosci.arizona.edu login:wcs password:nematode. Be warned that the response time using the NSFNET is slow at present. We look forward to receiving your gripes and annotations!