[
European Worm Meeting,
2006]
Jimmy Ouellet and Richard Roy. . The intestinal lineage of C. elegans provides an excellent model to examine how developmental cues control the cell cycle due to the invariant, developmentally-regulated cell cycle transitions characteristic of these cells. We have isolated 5 mutants that show irregularities in the control of intestinal nuclear division timing during post-embryonic development. We classified these mutants based on whether they possessed more intestinal nuclei (
rr33 and
rr45) or less intestinal nuclei (
rr42,
rr43 and
rr44) than wild-type. Since all these mutants hatch with the wild-type complement of intestinal cells they all affect the cell cycle transition from mitosis to karyokinesis and finally the onset of the endocycles that normally occurs at the first larval stage. By performing lineage analysis on
rr33 mutants we found that the intestinal nuclear divisions occur normally at the L1 stage, but they do not make the appropriate transition to endocycle that usually occurs at the L1 moult. Consequently they undergo an extra round of nuclear division early in the L2 stage and become tetranucleate. We mapped this
rr33 mutant close to
dpy-5 on LGI and we performed systemic RNAi with all the predicted genes in this interval to determine if any of these candidates could phenocopy
rr33. Only C32F10.2 (RNAi) phenocopied the
rr33 phenotype and this gene corresponds to
lin-35: the C. elegans orthologue of the Retinoblastoma(Rb) gene.
lin-35/Rb is part of the synMuv B complex, which is required for the proper regulation of vulva cell fates by inhibiting the ras signaling pathway. We found that all synMuv B components tested so far enhance the intestinal phenotype of
lin-35(
rr33), suggesting that the same complex that is required for vulva specification is also required for the appropriate regulation of intestinal nuclear divisions. The reiteration of a L1-specific division during the L2 stage (heterochronic phenotype) prompted us to look at some of the components of the miRNA as well as PTGS/RNAi pathway. We found that the
lin-35(
rr33); al! g-1
(gk214) double mutant is synthe! tically lethal, whereas neither of the single mutants show this phenotype suggesting that these two genes might function in a similar essential pathway or process in addition to their roles in regulating nuclear division. We also found that components of the endoRNAi pathway (
rrf-3 and
eri-3) suppress the intestinal phenotype of
lin-35(
rr33) suggesting that DCR-1 protein play a role in this transition and is limiting. When the endoRNAi pathway is abrogated DCR-1 becomes more available for the microRNA or exoRNAi pathway to restore the wild-type complement of intestinal division. We therefore conclude that LIN-35 and probably other components of the synMUV B are required for the timely cell cycle transition of the intestinal cells at the L1 stage and that
lin-35 cooperates with components of the PTGS and/or microRNA pathway to appropriately regulate this cell cycle transition. Distinct and Redundant Functions for C. elegans HP1 Proteins in Post-Embryonic Development. Palladino Francesca, Sonia Schott, Thomas Simonet, Vincent Coustham, Cecile Bedet, Marianthi Karali Laboratory of Molecular and Cellular Biology, UMR5161, Ecole Normale Superieure de Lyon, 69007 Lyon, France The highly conserved HP1 family proteins play an important role in the dynamic organization of nuclear architecture and in the epigenetic control of gene expression. Although most species contain more than one HP1 family member which differ in their chromosomal distribution, it is not known to what extent their function is redundant or specific in a developmental context. C. elegans has two HP1 homologues, HPL-1 and HPL-2. While
hpl-2 is required for the maintenance of a functional germline and for vulval development by acting in the Rb related synMuvB pathway, no obvious function has so far been attributed to HPL-1. We report the characterization of an
hpl-1 null allele. We show that while the absence of
hpl-1 alone results in no obvious phenotype,
hpl-1;
hpl-2 double mutants show synthetic, temperature sensitive phenotypes including larval lethality and severe defects in the development of the somatic gonad. Furthermore, we find that
hpl-1 has an unexpected role in vulval development by acting redundantly with
hpl-2, but not other genes previously implicated in vulval development. Localization studies show that like HPL-2, HPL-1 is a ubiquitously expressed nuclear protein. However, HPL-1 and HPL-2 show clear differences in their localization. This is most clearly evident in embryos, where HPL-1 and HPL-2 are found concentrated in non-overlapping nuclear foci. We find that the LIN-13 protein, which forms a complex with HPL-2, is absolutely required for recruitment of HPL-2, but not HPL-1, in these foci. Our results show that HPL-1 and HPL-2 play both unique and redundant functions in post-embryonic development. Altogether, these studies contribute to an understanding of the function of HP1 proteins in gene regulation throughout development, and in a more general way on how a general chromatin interacting protein may play specific roles in given developmental pathways.
[
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.