Intersex,a temperature-sensitive mutant of the nematode Caenorhabditis elegans

A temperature-sensitive mutation, isx-1(hc17), is reported in the nematode Caenorhabditis elegans which alters the sexual phenotypes of both genotypic sexes. At the restrictive temperature, XX animals are functionally female rather than hermaphroditic due to the absence of spermatogenesis, and XO animals develop as intersexes. These intersexes have normal male head and tail structures and exhibit some mating behavior, but possess hermaphrodite-like gonads which produce no sperm and usually contain a few oocytes. An abortive vulva is usually present and evidence is presented which suggests that the formation of the vulva by the hypodermis is induced by the underlying gonad. The direct effects of the mutation are confined to the descendants of four primordial gonad cells. Gametogenesis and gonad sheath development do not seem to be tightly coupled and are shown to differ in their responses to X-chromosome dosage. The interaction of the intersex mutation with mutant alleles of two transformer genes tra-1 and tra-2 is discussed and a model for the action of these genes in gonad development and sex determination is proposed.     

A methyl viologen-sensitive mutant of the nematode Caenorhabditis elegans

A methyl viologen (paraquat)-sensitive mutant, mev-1 (LG III), in Caenorhabditis elegans was about 4 times more sensitive to methyl viologen than the wild type. This mutant was also hypersensitive to oxygen. The brood size was about 1/4 that of the wild type. The average life span was determined to be 9.3 days as compared to 14.3 days for the wild type. The activity of superoxide dismutase (SOD), a scavenging enzyme for superoxide anion, was about half the wild-type level. We suggest that oxygen radicals may be involved in the normal aging mechanism in C. elegans.     

Cell contacts orient some cell division axes in the Caenorhabditis elegans embryo

Cells of the early Caenorhabditis elegans embryo divide in an invariant pattern. Here I show that the division axes of some early cells (EMS and E) are controlled by specific cell-cell contacts (EMS-P2 or E-P3 contact). Altering the orientation of contact between these cells alters the axis along which the mitotic spindle is established, and hence the orientation of cell division. Contact-dependent mitotic spindle orientation appears to work by establishing a site of the type described by Hyman and White (1987. J. Cell Biol. 105:2123-2135) in the cortex of the responding cell: one centrosome moves toward the site of cell-cell contact during centrosome rotation in both intact embryos and reoriented cell pairs. The effect is especially apparent when two donor cells are placed on one side of the responding cell: both centrosomes are "captured," pulling the nucleus to one side of the cell. No centrosome rotation occurs in the absence of cell-cell contact, nor in nocodazole-treated cell pairs. The results suggest that some of the cortical sites described by Hyman and White are established cell autonomously (in P1, P2, and P3), and some are established by cell-cell contact (in EMS and E). Additional evidence presented here suggests that in the EMS cell, contact-dependent spindle orientation ensures a cleavage plane that will partition developmental information, received by induction, to one of EMS's daughter cells.     

DNA glycosylase activities in the nematode,Caenorhabditis elegans

DNA glycosylases acting upon uracil- or 3-methyl-adenine-containing DNA have been detected in the sonic extracts of the nematode, Caenorhabditis elegans. 4 types of the asynchronously-growing worms, embryos obtained from gravid hermaphrodites, aseptically-hatched larvae, or dauer larvae. Uracil-DNA glcosylase activity was found in all 4 types of the extracts, and the activity was highest in the embryonic extract. In contrast, 3-methyladenine-DNA glycosylase activity was undetectable in the embryonic extract, while an equal level of activity was found in the other 3 types of the extracts. The results substantiate the ubiquity of base-excision repair in various organisms, and suggest that some of the repair functions may be developmentally regulated in multicellular animals.     

Noncanonical cell death programs in the nematode Caenorhabditis elegans

Genetic studies of the nematode Caenorhabditis elegans have uncovered four genes, egl-1 (BH3 only), ced-9 (Bcl-2 related), ced-4 (apoptosis protease activating factor-1), and ced-3 (caspase), which function in a linear pathway to promote developmental cell death in this organism. While this core pathway functions in many cells, recent studies suggest that additional regulators, acting on or in lieu of these core genes, can promote or inhibit the onset of cell death. Here, we discuss the evidence for these noncanonical mechanisms of C. elegans cell death control. We consider novel modes for regulating the core apoptosis genes, and describe a newly identified cell death pathway independent of all known C. elegans cell death genes. The existence of these noncanonical cell death programs suggests that organisms have evolved multiple ways to ensure appropriate cellular demise during development.     

Structural determinants underlying the temperature-sensitive nature of a Galpha mutant in asymmetric cell division of Caenorhabditis elegans

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Galpha(o)) and GPA-16 (Galpha(i)) are involved in generating forces that pull on astral microtubules and position the spindle asymmetrically. GPA-16 function has been analyzed in vivo owing notably to a temperature-sensitive allele gpa-16(it143), which, at the restrictive temperature, results in spindle orientation defects in early embryos. Here we identify the structural basis of gpa-16(it143), which encodes a point mutation (G202D) in the switch II region of GPA-16. Using Galpha(i1)(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Galpha. At the permissive temperature, the mutant Galpha was stable upon GTP binding, but switch II rearrangement was compromised, as were activation state-selective interactions with regulators involved in ACD, including GoLoco motifs, RGS proteins, and RIC-8. We solved the crystal structure of the mutant Galpha bound to GDP, which indicates a unique switch II conformation as well as steric constraints that suggest activated GPA-16(it143) is destabilized relative to wild type. Spindle severing in gpa-16(it143) embryos revealed that pulling forces are symmetric and markedly diminished at the restrictive temperature. Interestingly, pulling forces are asymmetric and generally similar in magnitude to wild type at the permissive temperature despite defects in the structure of GPA-16(it143). These normal pulling forces in gpa-16(it143) embryos at the permissive temperature were attributable to GOA-1 function, underscoring a complex interplay of Galpha subunit function in ACD.     

Gastrulation in the nematode Caenorhabditis elegans

Gastrulation in Caenorhabditis elegans has been described by following the movements of individual nuclei in living embryos by Nomarski microscopy. Gastrulation starts in the 26-cell stage when the two gut precursors, Ea and Ep, move into the blastocoele. The migration of Ea and Ep does not depend on interactions with specific neighboring cells and appears to rely on the earlier fate specification of the E lineage. In particular, the long cell cycle length of Ea and Ep appears important for gastrulation. Later in embryogenesis, the precursors to the germline, muscle and pharynx join the E descendants in the interior. As in other organisms, the movement of gastrulation permit novel cell contacts that are important for the specification of certain cell fates.     

Anaerobiosis in the nematode Caenorhabditis elegans

In Caenorhabditis elegans, mortality rates and changes in concentrations of carbohydrate stores and anaerobic end products were determined in anoxic (test) and normoxic (control) animals at two different temperatures (10 and 20 degrees C). The anoxic tolerance of the free-living nematode proved to be well-developed: at 10 degrees C, about 50% of animals had survived a period of 50 h of anoxia. The carbohydrate stores (approximately 30 mmol glycosyl units kg-1 freshweight (FW)) were reduced by two-thirds within 24 h of anoxia at both temperatures. L-lactate, acetate, succinate, and propionate were identified as the main anaerobic end products. The amounts and proportions of the end products were dependent on temperature. They did not accumulate very much in the tissues, but were mainly excreted. During anoxia, the metabolism of C. elegans was depressed to 3-4% of the aerobic value. The food-source Escherichia coli was found to be at least partly alive in the gut of the animals. To separate between anaerobiosis in animals and bacteria, cleaning procedures were applied, and additional control measurements were made: anaerobic end products produced either by E. coli alone or by bacteria-free (axenic) bred nematodes were quantified at identical incubation conditions.     

The embryonic cell lineage of the nematode Caenorhabditis elegans

The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.     

Ceramide glucosyltransferase of the nematode Caenorhabditis elegans is involved in oocyte formation and in early embryonic cell division

Ceramide glucosyltransferase (Ugcg) [uridine diphosphate (UDP)-glucose:N-acylsphingosine D-glucosyltransferase or UDP-glucose ceramide glucosyltransferase (GlcT): EC 2.4.1.80] catalyzes formation of glucosylceramide (GlcCer) from ceramide and UDP-glucose. There is only one Ugcg gene in the mouse genome, which is essential in embryogenesis and brain development. The nematode Caenorhabditis elegans has three Ugcg genes (cgt-1, cgt-2 and cgt-3), and double RNAi of the cgt-1 and cgt-3 genes results in lethality at the L1 larval stage. In this study, we isolated knockout worms for the three genes and characterized the gene functions. Each gene product showed active enzymatic activity when expressed in GM95 cells deficient in glycosphingolipids (GSLs). When each gene function was disrupted, the brood size of the animal markedly decreased, and abnormal oocytes and multinucleated embryos were formed. The CGT-3 protein had the highest Ugcg activity, and knockout of its gene resulted in the severest phenotype. When cgt-3 RNAi was performed on rrf-1 worms lacking somatic RNAi machinery but with intact germline RNAi machinery, a number of abnormal oocytes and multinucleated eggs were observed, although the somatic phenotype, i.e., L1 lethal effects of cgt-1/cgt-3 RNAi, was completely suppressed. Cell surface expression of GSLs and sphingomyelin, which are important components of membrane domains, was affected in the RNAi-treated embryos. In the embryos, an abnormality in cytokinesis was also observed. From these results, we concluded that the Ugcg gene is indispensable in the germline and that an ample supply of GlcCer is needed for oocytes and fertilized eggs to maintain normal membranes and to proceed through the normal cell cycle.     

Post-embryonic cell lineages of the nematode,Caenorhabditis elegans

The number of nongonadal nuclei in the free-living soil nematode Caenorhabditis elegans increases from about 550 in the newly hatched larva to about 810 in the mature hermaphrodite and to about 970 in the mature male. The pattern of cell divisions which leads to this increase is essentially invariant among individuals; rigidly determined cell lineages generate a fixed number of progeny cells of strictly specified fates. These lineages range in length from one to eight sequential divisions and lead to significant developmental changes in the neuronal, muscular, hypodermal, and digestive systems. Frequently, several blast cells follow the same asymmetric program of divisions; lineally equivalent progeny of such cells generally differentiate into functionally equivalent cells. We have determined these cell lineages by direct observation of the divisions, migrations, and deaths of individual cells in living nematodes. Many of the cell lineages are involved in sexual maturation. At hatching, the hermaphrodite and male are almost identical morphologically; by the adult stage, gross anatomical differences are obvious. Some of these sexual differences arise from blast cells whose division patterns are initially identical in the male and in the hermaphrodite but later diverge. In the hermaphrodite, these cells produce structures used in egg-laying and mating, whereas, in the male, they produce morphologically different structures which function before and during copulation. In addition, development of the male involves a number of lineages derived from cells which do not divide in the hermaphrodite. Similar postembryonic developmental events occur in other nematode species.     

Molecular genetics of cell death in the nematode Caenorhabditis elegans

In C. elegans, cell death can be readily studied at the cellular, genetic, and molecular levels. Two types of death have been characterized in this nematode: (1) programmed cell death, which occurs as a normal component in development; and (2) pathological cell death which occurs aberrantly as a consequence of mutation. Analysis of mutations that disrupt programmed cell death in various ways has defined a genetic pathway for programmed cell death which includes genes that perform such functions as the determination of which cells die, the execution of cell death, the engulfment of cell corpses, and the digestion of DNA from dead cells. Molecular analysis is providing insightinto the nature of the molecules that function in these aspects of programmed cell death. Characterization of some genes that mutate to induce abnormal cell death has defined a novel gene family called degenerins that encode putative membrane proteins. Dominant alleles of at least two degenerin genes, mec-4 and deg-1, can cause cellular swelling and late onset neurodegeneration of specific groups of cells. © 1992 John Wiley & Sons, Inc.     

Baculovirus p35 prevents developmentally programmed cell death and rescues a ced-9 mutant in the nematode Caenorhabditis elegans.

Programmed cell death, or apoptosis, occurs throughout the course of normal development in most animals and can also be elicited by a number of stimuli such as growth factor deprivation and viral infection. Certain morphological and biochemical characteristics of programmed cell death are similar among different tissues and species. During development of the nematode Caenorhabditis elegans, a single genetic pathway promotes the death of selected cells in a lineally fixed pattern. This pathway appears to be conserved among animal species. The baculovirus p35-encoding gene (p35) is an inhibitor of virus-induced apoptosis in insect cells. Here we demonstrate that expression of p35 in C. elegans prevents death of cells normally programmed to die. This suppression of developmentally programmed cell death results in appearance of extra surviving cells. Expression of p35 can rescue the embryonic lethality of a mutation in ced-9, an endogenous gene homologous to the mammalian apoptotic suppressor bcl-2, whose absence leads to ectopic cell deaths. These results support the hypothesis that viral infection can activate the same cell death pathway as is used during normal development and suggest that baculovirus p35 may act downstream or independently of ced-9 in this pathway.     

Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.     

Xenobiotic detoxification in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans is an important model organism for the study of such diverse aspects of animal physiology and behavior as embryonic development, chemoreception, and the genetic control of lifespan. Yet, even though the entire genome sequence of this organism was deposited into public databases several years ago, little is known about xenobiotic metabolism in C. elegans. In part, the paucity of detoxification information may be due to the plush life enjoyed by nematodes raised in the laboratory. In the wild, however, these animals experience a much greater array of chemical assaults. Living in the interstitial water of the soil, populations of C. elegans exhibit a boom and bust lifestyle characterized by prodigious predation of soil microbes punctuated by periods of dispersal as a non-developing alternative larval stage. During the booming periods of population expansion, these animals almost indiscriminately consume everything in their environment including any number of compounds from other animals, microorganisms, plants, and xenobiotics. Several recent studies have identified many genes encoding sensors and enzymes these nematodes may use in their xeno-coping strategies. Here, we will discuss these recent advances, as well as the efforts by our lab and others to utilize the genomic resources of the C. elegans system to elucidate this nematode's molecular defenses against toxins.     

Polyploid tissues in the nematode Caenorhabditis elegans

During larval development, the number of somatic nuclei in C. elegans hermaphrodites increases from 558 to 959 (J. E. Sulston and H. R. Horvitz, Dev. Biol. 56, 110-156, 1977; J. E. Sulston et al., Dev. Biol. 100, 64-119, 1983). At the same time, the animals increase about 60-fold in volume. We have measured the DNA contents of several classes of nuclei by quantitating the fluorescence of Hoescht 33258 stained DNA (D. G. Albertson et al., Dev. Biol. 63, 165-178, 1978). Probably all embryonic nuclei, including those of neurons, muscles, hypodermis, and intestine, are diploid at hatching. Neurons, muscles, and nondividing hypodermal nuclei remain diploid throughout larval development. The DNA content of the intestinal nuclei doubles at the end of each larval stage, reaching 32C by the adult stage. New hypodermal cells, generated by division of seam cells in the larval stages, undergo an additional round of DNA replication before fusing with the major syncytium (hyp7, Sulston et al., 1983). Thus the larval hyp7 syncytium comprises a fixed number of diploid embryonic nuclei plus an increasing number of tetraploid postembryonic nuclei. Some of the endoreduplications that occur in the intestinal and hypodermal lineages of C. elegans may correspond to nuclear or cellular divisions in another nematode Panagrellus redivivus (P. W. Sternberg and H. R. Horvitz, Dev. Biol. 93, 181-205, 1982).     

Molecular genetics of cell death in the nematode Caenorhabditis elegans.

In C. elegans, cell death can be readily studied at the cellular, genetic, and molecular levels. Two types of death have been characterized in this nematode: (1) programmed cell death, which occurs as a normal component in development; and (2) pathological cell death, which occurs aberrantly as a consequence of mutation. Analysis of mutations that disrupt programmed cell death in various ways has defined a genetic pathway for programmed cell death which includes genes that perform such functions as the determination of which cells die, the execution of cell death, the engulfment of cell corpses, and the digestion of DNA from dead cells. Molecular analysis is providing insight into the nature of the molecules that function in these aspects of programmed cell death. Characterization of some genes that mutate to induce abnormal cell death has defined a novel gene family called degenerins that encode putative membrane proteins. Dominant alleles of at least two degenerin genes, mec-4 and deg-1, can cause cellular swelling and late onset neurodegeneration of specific groups of cells.