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 in press) and not that of any of the other mutants so far isolated and examined (Waterston, unpublished).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.

Ouazana R (1980) Worm Breeder's Gazette "isolation and biochemical analysis of the cuticle collagen of C elegans, Bergerac strain."

Ouazana R

[Worm Breeder's Gazette, 1980]

The major components of the cuticle were found, in Ascaris des to be proteins (Bird 1956), the most important being collagen (Watson and Silvester 1959; Josse and Harrington 1964). In order to obtain this major protein, intact cuticles from C. elegans were isolated by sonication and incubation with 1% SDS, according to the method described by M. Kusch at the Woods-hole workshop. The purity of the preparation and the preservation of the in situ characteristics of the isolated cuticles were controlled by electron microscopy. Proteins represent about 65% of the dry weight of the isolated cuticles and collagen 70% of the proteins. Taking into account the fact that the cuticle is soluble in reducing solutions, as is the case for Ascaris (Evans et al. 1976), we prepared the C. elegans cuticle collagen by reduction with -mercaptoethanol in a buffer containing 8M Urea followed by carboxymethylation with sodium Iodoacetate. This collagen analyzed by SDS-Polyacrylamide gel electrophoresis gave seven bands (fig.). Their molecular weights are : 185000, 155000, 92000, 65000 (the predominant band) 51000, 32000, 20000 (the gamma, 11 and alpha1 components of Calf skin Type I Collagen were taken for calibration). All these components are sensitive to bacterial collagenase. The C. elegans cuticle collagen was submitted to molecular sieve chromatography and now to ion exchange chromatography on phosphocellulose to obtain purified components. [See Figure 1] The amino acid composition of the cuticular collagen of C. elegans and the fractions obtained by molecular sieve chromatography showed high hydroxyproline content (10%) and low proline content (13%) compared to Ascaris cuticle collagen (2% and 36% respectively, Evans et al. 1976). The proline and hydroxyproline contents are close to those found in vertebrate collagen. But like all known invertebrate cuticle collagen, C. elegans lacks hydroxylysine. The aim of the present work is to determine whether the separated components are genetically distinct chains and could represent different collagen types, or if these components all belong to the same type of collagen.

Itoh M et al. (1994) Worm Breeder's Gazette "Cloning of the cej-1 Gene Encoding a Novel Cell-Junction Protein in Caenorhabditis elegans M.L.A. Khan, M. Tabish, T. Fukushige, S. Tsukita, S. Tsukita,"

Itoh M, Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1994]

Intracellular tight junctions, such as zonula occludentes are known to form regulated barrier zones between both endothelial and the cells of epithelium, and provide a means to separate tissue compartments in higher animals. Recently a number of genes encoding "tight junction" proteins have been characterized from different species, such as the discs large tumor suppressor dlg gene of Drosophila (Woods & Bryant, 1991); human erythrocyte membrane protein p55 (Cho et al., 1992), PSD-95 from the rat brain synapses (Sadler et al, 1992), ZO-1 from human cells (Willot et al., 1993), and 220 kDa protein from mouse (Itoh et al., 1993). Although extremely different in the length of the primary sequence, a hallmark of these proteins is the presence of the SH3 and guanylate kinase domain that may not have the proper catalytic function. Using the monoclonal antibody T4192 raised against the mouse 220 KDa protein, we have screened a C. elegans cDNA expression library and cloned a cDNA that encodes a novel protein with no strong homology to the tight junction proteins, but shows the presence of the conserved amino acid residues in the SH3 and GK domains. The cDNA probe recognizes only one clone on the polytene filter, the Y50C3 ,that places the (cej-1 , cell junction -1)gene in the cluster on chromosome III near the mab-21 locus, in a region covered by three cosmid clones (F35G12 ,T23C9 ,and F1 OG11).Screening a genomic library, we have identified a positive clone corresponding to the cej-1 cDNA probe, and have subcloned a 9 kb genomic fragment that includes the gene. Southern blot analysis suggests that the cej-1 is a single copy gene, and the Northern blot data indicates that it encodes a mRNA of 1.5 kb. Immunocytochemical staining of the nematode on wholemount squash preparations using T4192 antibody which recognizes a 34 kDa protein on immunoblots, reveals that the antigen is present during embryogenesis at cell junctions both during the early and late embryonic development. In the larvae and adults, staining of the neural tissue and the excretory canal (H - Cell) is observed. We thank A. Coulson and other members of the Sanger Centre for their assistance in providing the YAC clones.

Waddle JA et al. (1994) Worm Breeder's Gazette "Acetone Extraction of Early C. elegans Embryos Reveals Persistent, Phalloidin-Stained Rings From Previous Cell Divisions"

Waddle JA, Waterston RH

[Worm Breeder's Gazette, 1994]

Recently, we fortuitously revealed phalloidin-stained, ring-shaped structures shared by sister cells in the early embryo. The detection of these structures requires formaldehyde fixation followed by 100% acetone extraction, a treatment that extracts other phalloidin-binding components including most of the cortical actin cytoskeleton in early nematode embryos. The time of appearance, position, morphology and number of phalloidin-positive rings within embryos suggest these organelles are persistent remnants from prior cell divisions. We refer to these rings as cell division remnants (CDRs). Preliminary characterization of these structures reveals the following: (i) A given embryo always has one fewer CDR than the total number of cells or a number equal to the number of cell divisions. (ii) The CDRs persist through many cell cycles and appear to be shared by all sister cells. (iii) The dimensions and position of the CDRs are similar to the initial size and location of the transient, CP-actin complexes that coincide with the onset of centrosome rotation in the P(1) lineage(1). For example, the CDR from first cleavage is located at a central site in the midfocal plane between AB and P(1) early in the 2-cell stage. Later, nuclear movements in the dividing AB cell push the CDR off-center in a manner which resembles the movement of the transient CP-actin complex during the 2-3 cell transition. (iv) The diameter of CDRs is variable (0.25-2.0 m) and unpredictable. In general, older CDRs are smaller but there is no obvious correlation between ring diameter and the cell cycle. (v) The CDR derived from first cleavage is invariably shared by EMS and ABa at the 4-cell stage, EMS and ABal at the 6-cell stage and MS and ABal at the 8-cell stage. At least some of the above observations suggest that once a CDR is formed, it is not divisible by subsequent cell divisions. Are CDRs involved in cell fate decisions? At the 4-cell stage, a signal from the EMS blastomere is required to instruct the anterior AB daughter to express a fate different from its sister, this interaction requires maternal expression of theglp-1 gene product (2,3). Theglp-1 protein is found in the membrane of both AB-derived cells at the 4-cell stage(4) in agreement with previous experiments which show these two cells have equivalent potential. Paradoxically, the EMS cell contacts both AB-derived cells at the 4-cell stage, yet only the anterior AB daughter expresses the EMS-induced fate change. How an EMS-derived signal is directed to the appropriateGlp-1 expressing AB descendant remains unclear. The invariant "path" of the persistent CDR from first cleavage to the 6-cell stage provides an internal, asymmetrically-positioned structure which could be exploited to distinguish between equivalent sites of contact between AB-derived cells and EMS at the 4 and 6-cell stages. The AB-EMS lineage interactions specify the dorsal-ventral(2) and left-right(5,6) embryonic axes at the 4 and 6-cell stages, respectively. We propose a model whereby the asymmetrically positioned CDR could be exploited to deliver a signal from EMS to specific AB descendants despite apparently equivalent contacts. A CDR model for the specification of the d-v embryonic axis in C. elegans. Fig 1. shows a schematic view of an experiment done by Priess and Thompson(2). At the 2-cell stage, segregation of determinants (shaded region) along the a-p axis in P(1) define the EMS and P(2) "ends" of the cell. Left unperturbed, embryos develop as shown on the left. On the right, the spindle in the AB cell is reoriented by physical manipulation with a blunt micropipette. One consequence of this manipulation is reorientation of the P(1) spindle. We propose that because the centrosome in P(1) is tethered to a CDR from first cleavage (bold circle between cells) the CDR, its associated cortex and determinants, and the anterior centrosome are repositioned as a unit. As a result, the absolute position of the EMS cell changes. At the stage, a signal from EMS is received by the AB daughter that shares the CDR from first cleavage; this interaction defines the d-v axis. A CDR model for the specification of the l-r embryonic axis in C. elegans. Fig. 2 shows a schematic view of an experiment done by Woods(5). EMS and its descendant MS are shaded to emphasize orientation. In all cases, anterior is left and ventral comes out of the page. In unoperated embryos, nuclear movements in the AB cells are skewed such that AB daughters on the left are slightly more anterior than their sisters on the right. If the skew of the AB spindles is reoriented prior to cytokinesis (right column), the l-r axis is switched giving rise to an enantiomeric embryo and adults. Reorientation of the AB cell positions after cytokinesis does not reverse the l-r axis(5). We propose the physical manipulation changes the absolute position of the CDR derived from first cleavage. This change might reroute an EMS-derived signal that makes ABal different from ABar and reverse the l-r axis. A correlation between the position of the CDR and cell fate specification can now be tested. The prediction is that the embryo manipulations described in Figs. 1 and 2 should invariably reposition the CDR upon successful reversal of the embryonic axes. We plan to test the models diagrammed in Figs. 1 and 2 and to investigate the composition of the CDRs using existing antibodies to various cytoskeletal components. REFERENCES: (l) Waddle et al, 1993 Worm Mtg, Abs#2. (2) Priess and Thompson, 1987 (3) Priess et al, 1987 (4) Kimble et al, 1992, CSH Symp Quant Biol 57:401 (5) Wood, 1991 Nature 349:536. (6) Schnabel, 1991, Mech Dev 34:85.

Edgar RS et al. (1977) Worm Breeder's Gazette "the nematode C elegans: a new organism for intensive biological study."

Wood WB, Edgar RS

[Worm Breeder's Gazette, 1977]

A small free-living soil nematode is receiving close scrutiny from a growing number of biological researchers. Some of these investigators believe that Caenorhabditis me the E. coli or at least the bacteriophage T4 of the animal world. C. elegans is a small transparent worm about a millimeter in length. Its genes are carried on five autosomes and a sex chromosome (X), and it has a genome size about 20 times that of E. coli. It generally reproduces as a self-fertilizing hermaphrodite (XX), but occasional males (XO), which arise by nondisjunction, permit sexual reproduction as well. The worm's prime virtues for research are its short life cycle (3 days) , ease of cultivation on E. coli as a food source, simple anatomy ( 810 somatic nuclei) and convenience for genetic analysis. Building upon the pioneering work of Dougherty (1), Nigon (2), and especially Brenner (3), who first described the animal's genetics, there are now more than a dozen laboratories in Canada, France, Germany, India, Japan, the United Kingdom, and the United States that are investing heavily in studies of this organism. From April 13 to April 16, researchers from these laboratories gathered for the first time to assess the status of their investment at a conference in Woods Hole, Massachusetts, supported in part by a grant from the National Institute of Aging, NIH. Many investigators initially turned to C. elegans in the hope of understanding its behavior in terms of its simple nervous system. The status of this work was a major topic at the meeting. C. elegans contains only 256 neurons, and a complete anatomical description of the nervous system at the electron microscope level is virtually at hand. Previous work had allowed detailed reconstruction of the head anterior sensory neuroanatomy, the ventral nerve cord, and the associated dorsal cord composed of processes from cells in the ventral cord (4). At the meeting, John White (MRC Cambridge) described reconstruction of the complex nerve ring that surrounds the pharynx and presumably plays the major role in processing sensory inputs to produce motor outputs. D. Hall (Albert Einstein College of Medicine) presented a reconstruction of the tail ganglia carried out in R. Russell's laboratory at Caltech. As a result of these studies, a complete wiring diagram for the whole nervous system of the animal is now within reach. Due to its small size, the system's function cannot yet be approached directly by standard electrophysiological techniques, and must await technical advances in electrode technology or in optical methods using potential-sensitive dyes. Meanwhile, however, Tony Stretton (University of Wisconsin) has shown that the anatomy of the related nematode Ascaris, which is sufficiently large for conventional electrophysiological studies, is virtually identical to that of C. elegans at least in the ventral cord. John Walrond from Stretton's laboratory presented evidence on which of the seven types of motor neurons in the ventral cord are inhibitory and which stimulatory. As a taste of things to come, R. Russell (now at University of Pittsburgh) presented a model to account for control of movement in C. elegans based on its ventral cord circuitry. More complete and detailed nervous system models should become possible as more functional features of the anatomy become understood. Another approach to obtaining such functional information is the investigation in several laboratories of nematode neurotransmitters. Earlier work by J. Sulston (MRC, Cambridge) localized three catecholamine-containing neurons, but mutants that did not produce catecholamines showed no demonstrable alteration in behavior (5). C. Johnson (University of Wisconsin) reported that in Ascaris acetylcholine appears to function as an excitatory transmitter, on grounds that it is synthesized in excitatory but not in inhibitory motor neurons. Johnson also reported on work carried out in R. Russell's laboratory at Caltech on the identification of a C. elegans mutant defective in acetylcholinesterase and another defective in choline acetyltransferase (the latter isolated in the laboratory of D. Hirsh, University of Colorado, as resistant to the drug trichlorofon). Jim Lewis (Columbia University) reported on the properties of putative acetylcholine receptor mutants isolated as resistant to the acetylcholine analog levamisole. R. Horvitz (MRC, Cambridge) reported on the behavior of mutants that fail to accumulate serotonin normally in certain neurons. The combination of biochemical, genetic, anatomical, and physiological approaches being taken by these researchers seems certain to provide insight into the processing of neuronal signals in C. elegans, and eventually could lead to an understanding of the animal's behavior at the level of its neuroanatomy and neurophysiology. Recently many investigators have recognized the potential of C. elegans for the study of development. The major portion of the meeting was devoted to description of the worm's development at the cellular level, and to studies of mutations that perturb it. The adult animal has only 810 somatic nuclei in six major cell types ( hypodermis, muscle, neurons, gut, gonadal sheath, and coelomocytes), and its anatomy at the cellular level is virtually invariant. A current major effort in the descriptive work has been the determination of cell lineages, and an important result of the meeting was the prospect that the complete cell lineage of C. elegans soon will be established from the zygote to the adult animal. Upon hatching from the egg, the juvenile or first-stage larva has only 540 somatic cells. During larval development, about 200 post- embryonic cells arise from division of a few blast cells present at hatching. J. Sulston and R. Horvitz (MRC, Cambridge) had previously traced in detail the origins of these post-embryonic cells (6). Their lineage studies revealed a remarkable reproducibility from one animal to another in times of cell division, paths of cell migrations, cell deaths, and ultimate differentiated cell fates. In experiments reminiscent of Boveri's classical observations on Ascaris embryogenesis, E. Schierenberg and other workers in G. von Ehrenstein's laboratory (Gottingen) now have established cell lineage in the egg up to the 186-cell embryo. Their studies, carried out with the light microscope using differential interference contrast optics and time lapse video recording reveal that characteristic rates of cell division are maintained in different subclones, regardless of cell migrations. The Gottingen group also has made detailed reconstructions of a 294-cell and 540-cell embryo from electron micrographs of serial sections. Surprisingly, the 540-cell stage is reached quite early in embryogenesis, about midway between fertilization and hatching and prior to most of the cell growth and differentiation that takes place before hatching, indicating that cell division and migration precede differentiation per se. Nevertheless, from the correspondence between the positions of cells in the 540-cell embryo and first stage larva, von Ehrenstein has been able to identify most of the embryonic cells with regard to their future fates. There is optimism that the remaining gap, between the 186-cell and the 540- cell stages can be filled by a combination of light and electron microscopy, to provide for the first time a complete description at the cellular level of the development of an animal from the egg to adulthood. The post-embryonic lineage of the gonad somatic cells, not investigated by Sulston and Horvitz, now has been determined by J. Kimble (University of Colorado). The gonad arises during larval development from a 4-celled precursor structure in both the hermaphrodite and the male. In the hermaphrodite the two somatic cells of the precursor give rise to a total of 142 cells, which form the gonadal sheath, spermatheca, and uterus. In the male a similar lineage produces about 50 cells to form the spermatheca and vas deferens. As in other tissues, the lineages are invariant in their significant features. The lineage in another single organ, the intestine, has been followed by K. Lew working in S. Ward's laboratory (Harvard Medical School), taking advantage of a mutant discovered by P. Babu (Tata Institute, Bombay; 7) in which gut cells fluoresce due to a defect in tryptophan catacolism. Lew has traced the lineage of the gut cells, which begin to fluoresce during embryogenesis, from a single precursor cell in the 8-cell embryo through the 20-cell juvenile gut to the adult 32-cell gut. He also has found aberrations in the lineage pattern in certain embryonic lethal mutants. On a related project, P. Siddiqui in P. Babu's laboratory reported that X irradiation of embryos heterozygous for such a fluorescence mutation gave rise to adults with fluorescent patches in the gut, suggesting an approach to the study of somatic crossing over in C. elegans.The factors responsible for lineage patterns are being investigated using a laser to selectively destroy certain cells, or using mutations that alter lineage. The preliminary results so far reported suggest that whereas there are some inductive or positional effects of cell differentiation, much of the developmental process is cell-autonomous. Two examples of nonautonomy were reported by J. White (MRC, Cambridge). Neuroblast cells that in a particular mutant fail to migrate to the normal position fail to differentiate completely into nerve cells. Also, if gonad development is prevented by laser ablation of the precursor cells, then the hypodermal cells that normally proliferate to form the vulva fail to do so, By contrast, other studies revealed a remarkable degree of cell autonomy. D. Albertson (MRC, Cambridge) reported on the behavior of certain blast cells that normally undergo a migration followed by a pattern of division to form 6 neurons and a hypodermal cell. In a particular mutant strain the blast cells undergo up to three abortive attempts at division to yield a polyploid cell, which differentiates to display cellular features of both hypodermal and nerve cells. R. Horvitz (MRC, Cambridge) reported on current progress in the genetic dissection of lineage patterns. He has isolated a large number of mutants that are defective in vulva formation and hence cannot lay eggs. These mutants so far define 15 different genes in which defects appear to directly affect the vulval lineage pattern, by preventing either divisions or normal migrations of precursor cells. Two laboratories are investigating differentiation of sperm, which are amoeboid cells in C. elegans. A large number of temperature- sensitive sperm-defective mutants have been isolated by D. Hirsh ( University of Colorado; 8), and S. Ward (Harvard Medical School), and analysis of these mutants is proceeding. At non-permissive temperature, some mutants appear to be blocked early in spermatogenesis and form no sperm, whereas others produce normal numbers of inactive sperm. All the mutants can lay viable eggs when mated with normal males. Ward reported that male sperm migrate rapidly to the hermaphrodite spermatheca following copulation and effectively supplant the endogenous sperm. Sperm may be required for some step in oogenesis, because some sperm-defective mutants fail to produce oocytes alone, but will do so when mated to males. The abundance of mutants and the prospects for isolating sperm in quantity make this system promising for studying the genetic control of a cellular differentiation pathway. Development of the male is virtually identical to that of the hermaphrodite until some hours after hatching, when a few of the post- embryonic cell lines display a male-specific pattern of division, migration, and differentiation. Intersex and transformer mutants defective in genes that control these processes were described by M. Klass (University of Colorado), Ward, and J. Brun (Lyon, France). These mutants should prove useful in determining how the male developmental pattern is superimposed upon that of the hermaphrodite. A special feature of the C. elegans life cycle has been studied by D. Riddle (University of Missouri). Normally, the young hatched larva progresses through a series of four molts to adulthood. In the absence of food, however, the second molt produces a special form, the dauer larva, which has an altered cuticle and can withstand adverse conditions (e.g., 4% SDS) and no food for periods up to 60 days. When presented with food, the dauer larva molts and continues the normal progress toward adulthood. Riddle has identified 7 genes whose functions are involved in the choice between the regular and the dauer developmental pathways. Mutants defective in these genes are constitutive dauer formers which enter the dauer pathway even in the presence of food. Some of these mutants have defects in sensory neuronal anatomy. Mutant analysis indicates that a larger number of genes probably is essential for recovery of the dauer larva and return to the standard developmental pathway. Brenner had estimated earlier that C. elegans carries about 2000 genes (3), somewhat fewer than Drosophila. If so, then more than 10% of them already have been identified. R. Horvitz (MRC, Cambridge) has undertaken the task of collating mapping data from different laboratories, and reported that over 200 genes have been identified and mapped with various degrees of precision. This number promises to increase rapidly, because investigators in the laboratories of R. Herman (University of Minnesota), D. Baillie (Simon Fraser University) , and W. Wood (Caltech) are embarking on exhaustive studies of lethals in defined regions of the genome. This work will be aided greatly by the availability of chromosomal duplications, deficiencies, and translocations, some of which already have been isolated and characterized in Herman's laboratory for use as crossover suppressors and balancers for lethals. Herman also reported on the isolation of stable tetraploid strains and on the instability of triploids generated by crosses to diploids. These studies incidentally provide information on the sex-determining mechanism in C. elegans, which as in Drosophila depends upon the balance between the numbers of sex chromosomes and autosomes. Promising studies also were reported on molecular genetic analysis of genes for muscle proteins. A large number of mutants defective in myosin, paramyosin (Q protein of the thick filaments) and other muscle components previously had been reported by R. Waterston and H. Epstein while working in Brenner's laboratory (9). At the meeting, Epstein (Stanford University) reported on the anatomical and biochemical properties of C. elegans muscle. At least 6 genes have been identified as being involved in muscle development. Of these, 2 appear to be the structural genes for myosin and paramyosin. Epstein reported that at least 2 different myosins exist in the nematode, one in the body wall muscle and another in the pharynx. The identified myosin gene controls the structure of the body wall muscle myosin. Some 30 alleles of this gene have been found, including a small internal deletion. S. MacCleod (MRC, Cambridge) reported on biochemical mapping of the mutational alterations in defective myosins by chemical cleavage and peptide analysis. Myosin and paramyosin mutations also have provided possible genetic access to the translational apparatus of C. elegans. R. Waterston ( Washington University) has shown that revertants of certain myosin mutations carry a second site suppressor that suppresses chain- terminating mutations in the myosin and paramyosin genes and certain alleles in genes with other diverse functions. This suppressor mutation is a promising candidate for the first tRNA nonsense suppressor to be described in a metazoan organism. C. elegans also is well suited to studies on the phenomenon of aging, in view of its short life cycle and the existence of a non- aging developmental variant, the dauer larva (10). M. Klass ( University of Colorado) reported on the effects of nutrition and other parameters on life span, and D. Mitchell (Boston Research Institute) presented evidence for entrainment of increased life span by prolonged cultivation under semi-starved conditions. However, attempts to find mutations that directly alter the life span so far have failed, and progress in this general area has been minimal. In a panel discussion following the session on aging the participants agreed that C. elegans has many potential advantages as an aging model, but that fruitful approaches exploiting genetics have yet to be developed. Many other studies were presented in addition to those mentioned. In this brief summary we have attempted only to highlight what in our opinions were the major themes of a rich and exciting meeting. The group of investigators who gathered at Woods Hole is taking a somewhat unusual holistic and cooperative approach to understanding the biology of a single organism. The meeting was invaluable in bringing together senior researchers and students with backgrounds in molecular, cellular, developmental, and neurobiology, in fostering the spirit of cooperative and integrated inquiry, and in generating a mutual enthusiasm that will help to make C. elegans an important model organism for intensive biological study during the next few years.